Late-Stage Activation of Toll-like receptor 3 Alleviates Cognitive Impairment and Neuropathology in an Alzheimer’s Disease Mouse Model

Abstract

This study was to investigate the effects of Toll-like receptor-3 (TLR3) activation on cognitive impairment and neuropathology in late-stage of Alzheimer’s disease in a mouse model. Amyloid protein precursor (APP)/presenilin-1 (PSEN1) (APP/PSEN1) mice were treated with Poly (I:C), a specific for TLR3. A panel of neurobehavioral tests were conducted to evaluate their cognitive functions. Aβ deposition, plasma Aβ levels, neuropathological changes, and activation of TLR3- TIR-domain-containing adapter-inducing interferon-β (TRIF) signaling were assessed by magnetic resonance imaging (MRI), electrophysiological recordings, transmission electron microscopy, Western blotting, immunofluorescence staining, and qPCR. The data demonstrated that Poly (I:C) significantly attenuated cognitive and neuropathological impairments, compared with APP/PSEN1 mice without Poly (I:C) treatment. Administration of Poly (I:C) significantly reduced brain Aβ_1-42 deposition and the levels of Aβ_1-40 and Aβ_1-42 in peripheral blood. In addition, treatment with Poly (I:C) significantly up-regulated the expression of anti-inflammatory factors and inhibited the expression of pro-inflammatory factors. The data indicated that systemic application of TLR3 agonist Poly(I:C) attenuated the brain damage, improved the cognitive function, and reduced the levels of Aβ_1-42 in brain and peripheral blood. The underlying mechanism might attribute to the up-regulation of p-IRF3 that increases the expression of anti-inflammatory factors and the inhibition of p-NF-κB that reduces the expression of pro-inflammatory factors. 

Supplementary Information

The online version contains supplementary material available at 10.1007/s12035-025-05092-0.

Background

Alzheimer’s disease (AD) is a chronic and progressive disease, and it the most common type of dementia [1]. Studies indicate that people age 65 and older survive an average of four to eight years after a diagnosis of Alzheimer’s dementia [2]. AD places heavy medical and social burdens on both patients’ families and society. Currently, β-amyloid (Aβ) deposition is considered one of the core pathological features of AD. Aβ is derived from the proteolytic cleavage of amyloid precursor protein (APP) by γ­secretases and β­secretases. Aβ is a heterogeneous group of peptides of varying lengths (ranging from 38 to 43 amino acids). Among them, Aβ_1–42 exhibits extremely strong cytotoxicity and neurotoxicity [3], which leads to myelin degeneration, axonal injury, neuronal death, inflammatory response, and, ultimately, cognitive dysfunction [4, 5]. Increasing evidence indicates that persistent immune inflammation in the brain is another core pathological change in AD [6]. Continuous Aβ stimulation in the brain results in the massive activation of glial cells, disrupting the balance between pro-inflammatory and anti-inflammatory processes in the brain, leading to chronic neuroinflammation [7]. Persistent pro-inflammatory responses not only lead to neuronal loss and neurodegeneration but also act as a link between the Aβ mechanism and the Tau protein and neurofibrillary tangles (Tau-NFTs) mechanism [8], exacerbating Aβ and NFTs accumulation [9].

Toll-like receptors (TLRs) belong to the type I transmembrane receptor superfamily, which are pattern recognition receptors in the innate immune system. They play a crucial role in initiating and regulating immune-inflammatory responses in AD. TLRs are activated by their ligands binding through its regulation protein and then activate their downstream protein kinases, causing the activation of transcription factor nuclear factor-kappa B (NFκB) and interferon regulatory factor (IRF) and ultimately inducing and regulating the release of immune inflammatory factors [10]. Unlike other TLRs, the TLR3 pathway is mediated by TRIF but not MyD88. TLR3 activation facilitates the activation of NFκB, IRF7, JNK, and p38MAPK, which in turn regulates the transcription of inflammatory cytokines [11, 12]. TLR3/TRIF-mediated signaling pathway activation can activate downstream protein kinases, leading to the activation of interferon regulatory factor 3 (IRF3) and IRF7, and then modulates the expression of type I interferon (IFN) [13]. In recent years, there has been increasing interest in the role of TLRs-mediated signaling pathways in the pathological changes observed in AD. TLRs signaling pathways play a significant role in the occurrence and development of AD. We have recently identified the crucial involvement of TLR2 in regulating neuroinflammation, Aβ deposition, and cognitive function in AD mouse brains. Other researchers also highlighted the indispensable roles of TLR4, TLR9, and TLR2 in AD processes [14–17]. Previous studies showed that the usage of TLR3 agonist Poly(I:C) can modulate innate immune responses, enhancing the resistance of neutropenic mice against Escherichia coli K1 meningoencephalitis [18]. Moreover, the pharmaceutical activation of TLR3/TRIF signaling inhibited the expression of pro-inflammatory factors, promoting the recovery of neural function after a stroke [19]. However, there have been fewer reports on the specific role of TLR3 in AD.

In this study, 15-month-old APP/PSEN1 mice were treated with TLR3 agonist Poly(I:C). Aβ deposition, white matter injury, grey matter injury, cognitive functions, and potential mechanisms were investigated. 

Methods

Animals

Male APPswe/PSEN1 dE9 transgenic (AD, App + Psen1 +) mice and male C57BL/6 J wild-type (WT) mice were purchased from the GemPharmatech (China). All mice were housed in the Animal Breeding Center of Xuzhou Medical University at a temperature of 21–23 °C with a 12-h light–dark cycle. The mice had free access to food and water ad libitum. All the experimental mice were randomly assigned to four groups to minimize potential bias: (1) saline-treated WT mice, (2) Poly(I:C)-treated WT mice, (3) saline-treated AD mice, and (4) Poly(I:C)-treated AD mice.

Drug Administration

Poly(I:C) was obtained from Tocris Bioscience (Minneapolis, MN, USA) and was dissolved in 0.9% NaCl to the concentration of 1 mg/ml. This solution was stored at − 20 °C. In the Poly(I:C) treatment groups, mice were intraperitoneally injected with Poly(I:C) at a dosage of 5 mg/kg body weight every 4 days for a total duration of 3 months starting from 12 months of age and continuing until they reached 15 months of age. Mice in the saline control groups were injected with the same volume of normal saline (0.9%NaCl). Behavioral tests were conducted after the three-month treatment. No significant disease-like behaviors were observed in these groups after injection. In addition, during behavioral testing, continuous injections were also scheduled every 4 days to ensure continuous exposure to Poly(I:C).

Morris Water Maze Test

Morris water maze tests were conducted to assess learning and memory abilities, as described previously [20]. Briefly, after systematic injection of Poly (I:C), place navigation tests were conducted. The experimental mice were gently placed into different quadrants of the pool to find the hidden platform. The mice were allowed to stay on the platform for 10 s if they found the platform within 60 s. If the mice could not find the platform, they were guided to the platform and could stay for 10 s. The mice were placed in different quadrants each day during the seven consecutive days of training. Subsequently, a place probe test was conducted. On the eighth day, the platform was removed. The mice were gently placed into the pool and allowed to swim freely for 60 s. The time (latency time) it took to find the platform, average speed, the number of times the mice crossed the platform and the time spent in the target quadrant were recorded.

Magnetic Resonance Imaging (MRI)

MRI scans were performed to evaluate white matter damage and brain atrophy, as we described previously [21, 22]. The MRI scans included DTI (diffusion tensor imaging) and T2 WI (T2-weighted imaging) and were performed using a Varian 7.0 T/160 mm magnetic resonance instrument (Varian Palo Alto, CA, USA). For the T2 WI scans, the following parameters were used: TR (repetition time) = 2500 ms; TE (echo time) = 60 ms; ETL (echo train length) = 8; FOV (field of view) = 20 mm * 20 mm; slice thickness/gap = 0.8 mm/1.0 mm; matrix = 256 * 512. For the DTI scans, the following parameters were used: TR = 2000 ms; TE = 36 ms; and the gradient directions [Gx, Gy, Gz] were [1, 1, 0], [1, 0, 1], [0, 1, 1], [− 1, 1, 0], [0, − 1, 1], and [1, 0, − 1]. The b-value was 2500 s/mm²; FOV = 20 mm * 20 mm; slice thickness = 0.5 mm; and matrix = 192 * 192. Following the data acquisition, ParaVision 6.0.1 software was employed to reconstruct the T2 WI images. Subsequently, Image J software was utilized to analyze cortical thickness and hippocampal volume. Anisotropy parameters, specifically fractional anisotropy (FA) and axial diffusion (DA), were analyzed using DSI Studio software.

Patch Clamp Electrophysiological Recordings

Mice were anesthetized and perfused with ice-cold sucrose-rich slicing solution (SRSS) containing (in mM) 75 sucrose, 85 NaCl, 2.5 KCl, 1.25 NaH₂PO₄, 25 NaHCO₃, 4 MgCl2, 0.5 CaCl₂, and 25 glucoses. Hippocampal slices (300 μm) were prepared and stored in artificial cerebrospinal fluid (ACSF) for subsequent recording. All solutions were oxygenated with 95% O₂ and 5% CO₂. Slices were transferred to the recording chamber, perfused with oxygenated ACSF, and field excitatory postsynaptic potentials (fEPSPs) were recorded. Input–output (I/O) curves were generated by gradually increasing the stimulation intensities (0.05 to 0.35 mA). Paired-pulse ratios (PPR) were calculated. Long-term potentiation (LTP) was induced using a high-frequency stimulation (HFS) protocol, and the fEPSPs were recorded and analyzed using MultiClamp 700B and software provided by Molecular Devices.

Transmission Electron Microscopy (TEM)

To observe the structure of the myelin sheath in the corpus callosum (CC) and external capsule (EC) region, we performed a TEM analysis following our previously established protocol [21]. Mice were sacrificed after behavioral testing, and their brains were perfused. The CC/EC region was dissected into 1-mm³ cubes, fixed using an electron microscope fixative solution, sectioned using an ultrathin microtome (NULL A-1170), and then imaged with an electron microscope (FEI Tecnai G2 Spirit Twin). In each group, 125 axons were measured using Image J software. The relationship between the thickness of the myelin sheath, the g-ratio of individual fibers, and the diameter of their respective axons (shown as a scatter plot) was calculated and analyzed.

Crystal Violet (CV) Staining

To visualize neuronal loss in the hippocampus and cortex, we performed CV staining according to our previous reports [21]. Slices were placed into xylene, absolute ethanol, 95% alcohol, 80% alcohol, 70% alcohol, distilled water, CV solution, and finally decolorized with 70% ethanol. In the cortex and hippocampal regions (CA1), we used a microscope (Leica DM2700M) to count the number of intact neurons in the brain tissue.

Western Blot

Western blotting was performed as we described previously [23]. Proteins were extracted and separated using an SDS-PAGE system and then transferred onto 0.45-μm polyvinylidene difluoride (PVDF) membranes (Millipore IPVH00010, MA, USA). Subsequently, the PVDF membranes were incubated overnight at 4 °C with primary antibodies. After washing with buffer, the PVDF membrane was further incubated with peroxidase-conjugated secondary antibodies. The signal was detected using the high-sensitivity ECL Western Blotting Substrate (Tanon, Shanghai, China) and imaged using the Bio-Rad ChemiDoc imaging system (Bio-Rad Inc, USA). Primary antibody dilution and vendor information for the western blotting and IF staining protocols are presented in Table 1.

Table 1.

List of primary antibodies used in the present study

Antibodies	Host species	WB dilution	IF staining dilution	Companies	Catalog#
Aβ 1–42	Rabbit	0.388889	0.25	Proteintech	25,524AQ–1-AP
Iba-1	Goat	0.388889	0.388889	Abcam	Ab5076
Iba-1	Rabbit	0.388889	N/A	Wako	019–19741
GFAP	Mouse	N/A	0.736111	Sigma	G3893
GFAP	Rabbit	0.388889	N/A	Abcam	ab7260
Neun	Mouse	N/A	0.388889	EMD Millipore	MAB377
MBP	Rabbit	0.736111	0.736111	Abcam	ab218011
IRF3	Rabbit	0.388889	N/A	Bioss	2993R
p-IRF3	Rabbit	0.388889	0.388889	Bioss	9278R
NF-κB p65	Rabbit	0.388889	N/A	Cell Signaling	#8242
p-NF-κB p65	Rabbit	0.388889	0.388889	Cell Signaling	#3033

Immunofluorescence (IF) Staining

IF staining was performed as we described previously [24]. Briefly, brain slices (thickness: 25 µm) were taken out of the refrigerator (− 20 °C), placed at room temperature for 30 min for rewarming, washed three times with 0.3% phosphate-buffered saline with Tween detergent (PBST) (5 min each time), washed once with 1% PBST for 15 min, washed with 0.3% PBST three times (5 min each time), and then blocked at room temperature with 1% goat serum for one hour. After the blocking was completed, the primary antibody was added in proportion to 1% goat serum and placed in a 4 °C refrigerator for overnight incubation, followed by secondary antibodies accordingly.

Quantitative Polymerase Chain Reaction (qPCR)

qPCR was performed as described previously [24]. Total RNA was extracted from mouse brain tissues using Trizol reagent. The extracted total RNA was then reverse-transcribed into cDNA using PrimeScript™ RT Master Mix (Takara, Code No. RR360 A). Q-PCR was performed on the CFX96™ Real-Time PCR Detection System (Bio-Rad) using SYBR® Premix Ex Taq TM II (Tli RNaseH Plus) (Takara, #RR820 A). RNA quantification was based on the 2^−ΔΔCt method. The housekeeping gene used for normalization in our qPCR experiments was β-actin. The specific primers used for the PCR reaction in this study are listed in Table 2.

Table 2.

List of primers used in the present study

Target genes	Forward	Reverse	Amplicon length (bp)	Annealingtemperature
IL-1β	5′- GCAACTGTTCCTGAACTCAACT-3′	5′-ATCTTTTGGGGTCCGTCAACT-3′	621	64
TNF-α	5′-GACGTGGAACTGGCAGAAGAG-3′	5′-TTGGTGGTTTGTGAGTGTGAG-3′	228	63.7
IL-6	5′- TGGTCTTCTGGAGTACCATAGC-3′	5′- TGTGACTCCAGCTTATCTCTTGG-3′	140	64.1
IFN-β	5′-CAGCTCCAAGAAAGGACGAAC-3′	5′-GGCAGTGTAACTCTTCTGCAT-3′	138	63.4
β-actin	5′-GGCTGTATTCCCCTCCATCG-3′	5′-CCAGTTGGTAACAATGCCATGT-3′	241	64.5

Enzyme-Linked Immunosorbent Assay (ELISA)

ELISA was performed as described previously [25]. Briefly, the standard protein curve was prepared firstly with known concentrations. Then, 10 μl of the plasma samples were added to designated wells of 96 wells plate and incubated for two hours at room temperature with gentle continual shaking (~ 500 rpm). The plates were incubated with the first antibody after washing, and then incubated with corresponding second antibodies after washing. The chromogenic solution was added after washing. The absorbance at 450 nm was measured within 30 min of adding stop solution in a microplate reader. The results were calculated using a log–log curve fitting.

Statistical Analyses

All experimental data are presented as mean ± SEM (standard error of the mean). The data were processed and statistical graphs were generated using GraphPad Prism 8.0 software.

The normality of the data was assessed using the Shapiro–Wilk normality test and the homogeneity of variance was assed using the Brown–Forsythe test. For data that passed the normality test and showed homogeneity of variance, one-way or two-way ANOVA was used for analysis. When the data exhibited homogeneity of variance, pairwise comparisons were performed using the Tukey post hoc test; when the data did not have homogeneous variance, the Dunnett T3 post hoc test was used. Additionally, for correlation analysis between variables, two-tailed Pearson correlation analyses were performed. Statistical significance was considered when p ≤ 0.05.

To ensure adequate statistical power for our experiments, we have conducted post hoc power analyses using both the FTestAnovaPower module in Python (Statsmodels) and G*Power software based on the actual group means and variances observed in our ELISA results (Aβ42 and Aβ40). The calculated effect sizes indicated a strong difference among groups. Under the standard settings of α = 0.05 and power = 0.80, the estimated minimum required sample size per group was approximately 3. Furthermore, we applied the same power analysis approach to the results of Western blot and qPCR in the present study based on the observed group means, variances, and sample numbers. The results confirmed that under the standard assumptions of a significance level (α) of 0.05, all the powers were larger than 0.80.

Results

Intraperitoneal Injection of Poly (I:C) Improves Cognitive Function in AD Mice

To investigate the potential therapeutic effects of Poly(I:C) on AD mice, Poly (I:C) or saline were intraperitoneally injected into AD and WT mice (5 mg/kg, once every 3 days), and a Morris water maze test was conducted to examine their learning and memory ability.

Both WT and AD mice were intraperitoneally injected with 5 mg/kg of Poly (I:C) or saline for 3 months, and their cognitive functions were examined using the Morris water maze (MWM). (A) Swimming track plots for the MWM test. (B) Latency time to find the hidden platform (two-way ANOVA and Tukey’s test). (C) Time spent in the target quadrant (Kruskal–Wallis test and Dunn’s test). (D) Platform crossing frequency (Kruskal–Wallis test and Dunn’s test). (E) Total distance travelled in the target quadrant (Kruskal–Wallis test and Dunn’s test). (F) Swimming speed in the MWM test (one-way ANOVA & Tukey’s test). Data are present as mean ± SEM, n = 11–15/group. *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001 versus AD + Saline group.

In the positioning cruise experiment of the Morris water maze (MWM) test, the results revealed that compared to saline-treated WT mice, saline-treated AD mice took significantly longer to locate the platform. In contrast, Poly (I:C)-treated AD mice required significantly less time to find the platform than saline-treated AD mice (Fig. 1A, B). In the space exploration of the MWM test, saline-treated AD mice spent significantly less time in the target quadrant than saline-treated WT mice. However, Poly (I:C)-treated AD mice spent significantly more time in the target quadrant than saline-treated AD mice (Fig. 1C). There were no significant differences in the swimming speeds among the mice in each group, and the prompt mouse MWM test results were unaffected by swimming speed (Fig. 1F). The total distance travelled by the saline-treated AD mice in the target quadrant was significantly shorter than that travelled by the saline-treated WT mice, but Poly (I:C)-treated AD mice moved significantly further in the target quadrant than saline-treated AD mice (Fig. 1E). These results suggest that intraperitoneal injection of Poly (I:C) significantly improves the cognitive function of AD mice.

Fig. 1.

Poly (I:C) treatment improves the recovery of cognitive function in the AD mouse model

Intraperitoneal Injection of Poly (I:C) Reduces the Deposition of Aβ_1–42 in AD Mouse Brains

Aβ-induced neurotoxicity is considered a key factor in the formation and progression of AD [26]. We further examined the expression of the major toxic isoform Aβ_1–42in AD mouse brains using immunofluorescence staining and western blotting. Our Aβ_1–42 immunofluorescence staining results showed no obvious positive staining of Aβ_1–42 in the brain of WT mice. In contrast, the deposition of Aβ_1–42 in the hippocampus and cortex of Poly (I:C)-treated AD mice was significantly reduced compared to that of saline-treated AD mice (Fig. 2D). Western blotting further confirmed this finding (Fig. 2A). Based on our results, in comparison to saline-treated WT mice, the expression of Aβ_1–42 protein in the cerebral cortex and hippocampus of saline-treated AD mice was significantly increased. However, the expression of Aβ_1–42 protein in the cerebral cortex and hippocampus of Poly (I:C)-treated AD mice was significantly lower than that in saline-treated AD mice (Fig. 2B, C). These results suggest that intraperitoneal injection of Poly (I:C) significantly reduced the deposition of Aβ_1–42 in the cerebral cortex and hippocampus of AD mice.

Fig. 2.

Poly(I:C) treatment reduces the deposition of Aβ_1–42 in the brain and the levels of Aβ_1–40 and Aβ_1–4 in the plasma of the AD mouse model

After the behavioral tests, western blotting and immunofluorescence staining studies were used to assess Aβ_1–42 deposition in the mouse brain. Plasma Aβ_1–40 and Aβ_1–42 levels were determined by ELISA. (A) Western blot bands of Aβ_1–42 in the cerebral cortex and hippocampus. (B–C) Quantitative analysis of protein expression of Aβ_1–42 in the cortex and hippocampus. (D) Representative pictures of Aβ_1–42 immunofluorescence staining in the brains of mice in each group. (E) Aβ_1–42 content in plasma. (F) Aβ_1–40 content in plasma. Data are presented as mean ±SEM, n= 4–5/group. The statistical analysis included a one-way ANOVA and Tukey’s test. *p≤ 0.05, **p≤ 0.01, ***p≤ 0.001 versus AD + Saline group.

Intraperitoneal Injection of Poly (I:C) Reduces both Aβ_1–40 and Aβ_1–42 Levels in the Plasma of AD Mice

We also measured the Aβ protein content in the peripheral blood plasma of mice. The results showed that the plasma Aβ_1–42 content in saline-treated AD mice was significantly higher than that in saline-treated WT mice, while the Aβ_1–42 content in Poly(I:C)-treated AD mice was significantly lower than that in saline-treated AD mice (Fig. 2E). The plasma Aβ_1–40 content in saline-treated AD mice was significantly higher than that in saline-treated WT mice, while the Aβ_1–40 content in Poly(I:C)-treated AD mice was significantly lower than that in saline-treated AD mice (Fig. 2F). These results indicate that intraperitoneal injection of Poly(I:C) significantly reduces the levels of plasma Aβ_1–40 and Aβ_1–42 in AD mice.

Intraperitoneal Delivery of Poly(I:C) Attenuates White Matter Damage in AD Mice

White matter plays a crucial role in cognitive function [27]. Thus, cerebral white matter integrity was examined following Poly (I:C) treatment in the AD mice. First, 9.4 T MRI was performed using Diffusion tensor imaging (DTI). The FA images were reconstructed using DSI studio software. The FA and DA values in the external capsule (EC) were also quantified using DSI Studio.

Our results showed that the DA and FA values in saline-treated AD mice were significantly lower than those in saline-treated WT mice. In contrast, the DA and FA values in Poly (I:C)-treated AD mice were significantly higher than those in saline-treated AD mice (Fig. 3A, B, and C). We also conducted correlation analyses between FA values, DA values, and performance metrics from the MWM test, including latency to platform, time spent in the target quadrant, and platform crossing frequency, in both saline-treated AD and Poly(I:C)-treated AD mice. As shown in Fig. 3D–I, FA and DA values were positively correlated with latency to platform and time spent in the target quadrant. FA and DA values were negatively correlated with latency to platform, suggesting that intact white matter (WM) is vital to cognitive function in AD mice.

Fig. 3.

Poly(I:C) treatment attenuates white matter damage in the AD mice

After the behavioral tests, MRI, TEM, MBP immunofluorescence staining, and western blotting were used to evaluate the white matter injury of mice. (A) Representative DTI axial views of FA maps (red arrows: measurement site). (B–C) Quantitative analysis of DA and FA values in the EC. (n = 5/group, one-way ANOVA and Tukey’s test). (D–I) The correlation analysis of latency to escape, platform cross frequency, time spent in target quadrant in the MWM test and FA values, DA values in the EC areas (n = 5/group, Pearson correlation analysis). (J) Representative TEM images showing the ultrastructure of axons and myelin sheaths. Quantitative analysis of the (K) g-ratio and (L) myelin thickness (n = 125 axons/group, simple linear regression and slope comparison). (M) Representative pictures of MBP immunofluorescence staining in the brains of mice in each group. (N–O) Western blot bands and quantitative analysis of MBP in the cerebral cortex and hippocampus. Data are presented as mean ± SEM, n = 4/group. The statistical analysis included a one-way ANOVA and Tukey’s test. *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001 versus AD + saline group.

Furthermore, we examined the ultrastructure of myelin using TEM. In comparison to saline-treated WT mice, saline-treated AD mice exhibited an increased g-ratio and decreased myelin sheath thickness (Fig. 3J–L), However, the g-ratio in Poly(I:C)-treated AD mice was significantly lower than that in saline-treated AD mice, and the thickness of the myelin sheath was significantly greater than that in saline-treated AD mice (Fig. 3J–L). Subsequently, we utilized immunofluorescence staining and western blotting to assess the expression of myelin basic protein (MBP), a myelin marker, in the cerebral cortex and hippocampus. The MBP immunofluorescence staining results revealed that the expression of MBP in the cerebral cortex and hippocampus of saline-treated AD mice was significantly lower than that in saline-treated WT mice. However, the expression of MBP in the cerebral cortex and hippocampus of Poly(I:C)-treated AD mice was significantly higher than that in saline-treated AD mice (Fig. 3M). This finding was consistent with a subsequent quantitative analysis using western blotting (Fig. 3N, 7O). In summary, these results suggest that intraperitoneal injection of Poly(I:C) is beneficial for repairing white matter damage in the AD brain.

Fig. 7.

Poly (I:C) treatment increases the expression of p-IRF3 and decreases the expression of p-NFκB in astrocytes in the brain in the AD mice model

Systematic Application of Poly(I:C) Attenuates Brain Atrophy and Neuronal Loss in AD Mice

Brain atrophy and neuronal loss represent the most critical pathological changes in AD. Such changes can contribute to cognitive impairment and memory loss [28]. In our study, we utilized T2 WI images to assess cortical thickness and hippocampal volume. Neuronal loss in the mouse cerebral cortex and hippocampus was detected using cresyl violet (CV) histological staining. The statistical analysis of T2 WI images revealed that the cerebral cortex in saline-treated AD mice exhibited a significantly reduced thickness compared to saline-treated WT mice. However, the cortical thickness in Poly(I:C)-treated AD mice was markedly increased compared to their saline-treated counterparts (Fig. 4B). Additionally, the hippocampal volume was notably decreased in saline-treated AD mice when compared to saline-treated WT mice. Conversely, the hippocampal volume in Poly(I:C)-treated AD mice was significantly larger than that in saline-treated AD mice (Fig. 4C).

Fig. 4.

Poly (I:C) treatment attenuates cortical hippocampus atrophy and neuronal loss in the AD mouse model

After the behavioral tests, cortical thickness and hippocampus volume were measured using MRI T2 WI images to evaluate brain atrophy. Neuronal loss in the cortex and hippocampus was determined by CV staining. (A) Representative image of hippocampus and cortex in T2 WI of MRI. (B) Quantitative analysis of cortical thickness. (C) Quantitative analysis of hippocampus volume. (D) Representative pictures of Nissl staining in the cortex and hippocampus CA1. (E–F) Quantitative analysis of neurons with normal morphology in the cerebral cortex and hippocampus CA1. Data are present as mean ± SEM, n = 5–6/group. The statistical analysis included a one-way ANOVA and Tukey’s test. *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001 versus AD + Saline group.

The CV staining results demonstrated a significant decrease in the number of neurons in the cerebral cortex hippocampal CA1 area of saline-treated AD mice when compared to saline-treated WT mice. Conversely, the number of neurons in the cerebral cortex CA1 region of the hippocampus in Poly(I:C)-treated AD mice was significantly higher than that in saline-treated AD mice (Fig. 4E, F). In conclusion, these results suggest that intraperitoneal injection of Poly(I:C) can reduce neuronal loss in the AD brain, thereby attenuating cortical and hippocampal atrophy.

Systematic Application of Poly(I:C) Improves Synaptic Function in AD Mice

Next, we examined the effect of Poly(I:C) treatment on the maintenance of LTP, a form of synaptic plasticity that is widely thought to be the basis of learning and memory in AD mice. Electrophysiological recordings in hippocampal slices showed that HFS-induced LTP of fEPSPs was significantly inhibited in saline-treated AD mice but not in AD mice treated with Poly(I:C) (Fig. 5B and C). To determine whether there were presynaptic changes in these mice, we measured the PPR of the fEPSPs. The PPR showed a significant increase, with 50 ms intervals in saline-treated AD mice, but Poly(I:C)-treated AD mice did not show this effect (Fig. 5D). To examine the effect of Poly(I:C) treatment on synaptic transmission, the input–output curves of fEPSPs were also recorded. The input–output curves decreased in saline-treated AD mice, but Poly (I:C) treatment markedly improved synaptic transmission in AD mice (Fig. 5E). However, Poly (I:C) treatment did not change the LTP, PPR, and input–output curves of fEPSPs in WT mice. Taken together, these results suggest that intraperitoneal injection of Poly (I:C) can alleviate synaptic impairments in AD mice.

Fig. 5.

Poly (I:C) treatment ameliorates impairments in synaptic function in the AD mouse model

(A) Schematic diagram of the measurement location. (B) Representative traces of fEPSP and time course of the fEPSP slope during LTP recording. Scale bars represent 0.4 mV and 10 ms. (C) Quantitative analysis of the normalized average fEPSP slopes during the last 10 min (n = 8; 3 mice per group, one-way ANOVA and Tukey’s test). (D) Representative traces and quantitative analyses of the PPR from 4 groups (n = 11; 3 mice per group, one-way ANOVA and Tukey’s test). (E) Representative traces and quantitative analyses of I/O (n = 12, 10, 11, and 11; 3 mice per group, two-way ANOVA and Tukey’s test). Data are presented as mean ± SEM, *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001 versus AD + Saline group.

Systematic Application of Poly (I:C) Inhibits the Activation of Microglia and Astrocytes in AD Mouse Brains

The excessive proliferation and activation of glial cells has been shown to significantly aggravate the pathological process of AD [20]. In the present study, the Western blots was used to evaluate the markers for microglia (Iba-1) and astrocytes (GFAP) in the tissues from cerebral cortex and hippocampus of mice. As depicted in Fig. 6A, C, and 16D, the levels of Iba-1 protein were elevated in the brains of saline-treated AD mice compared to WT controls. Interestingly, in Poly(I:C)-treated AD mice, the levels of Iba-1 in the hippocampus and cortex were significantly lower than those in saline-treated AD mice. Western blot analysis also revealed that the expression of GFAP protein in the cerebral cortex and hippocampus of saline-treated AD mice was significantly higher than that in saline-treated WT mice. Interestingly, in Poly(I:C)-treated AD mice, the expression of GFAP protein in the cerebral cortex and hippocampus was significantly lower than that in saline-treated AD mice (Fig. 6B, E, and F). These results strongly indicate that intraperitoneal injection of Poly(I:C) substantially reduces the proliferation and activation of astrocytes and microglia in the AD brain.

Fig. 6.

Poly (I:C) treatment inhibited the activation of microglia and astrocytes in the brain in the AD mouse model

(A) Western blot bands of Iba-1 in the cerebral cortex and hippocampus. (B) Western blot bands of GFAP in the cerebral cortex and hippocampus. (C–D) Quantitative analysis of Iba-1 protein expression in the cortex and hippocampus. (E–F) Quantitative analysis of GFAP protein expression in the cortex and hippocampus. Data are presented as mean ± SEM, n = 4/group. The statistical analysis included a one-way ANOVA and Tukey’s test. *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001 versus AD + Saline group.

The Cellular Distribution of p-IRF3 and p-NFκB in AD Mouse Brains

In this study, p-IRF3 and p-NFκB were co-stained with neuron (NeuN), microglia (Iba-1), and astrocytes (GFAP) in AD mice.

(A) Representative immunofluorescence staining for p-IRF3 and GFAP in the cortex. (B) Representative immunofluorescence staining for p-IRF3 and GFAP in the hippocampus. (C–D) Quantitative analysis of p-IRF3 and GFAP immunofluorescence staining in the cortex and hippocampus. (E) Representative immunofluorescence staining of p-NFκB and GFAP in the cortex. (F) Representative immunofluorescence staining of p-NFκB and GFAP in the hippocampus. (G–H) Quantitative analysis of p-NFκB and GFAP immunofluorescence staining in the cortex and hippocampus. Data are presented as mean ± SEM, n = 4/group. The statistical analysis included a one-way ANOVA and Tukey’s test. *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001 versus AD + Saline group.

The results showed that p-IRF3 and p-NFκB were significantly expressed in astrocytes, while their expression in neurons and microglia was not prominent (Fig. 7A, B, E, F). The immunofluorescence results showed that in astrocytes, the expression levels of p-IRF3 in the Poly (I:C)-treated WT mice were higher than those in saline-treated WT mice in the hippocampus (Fig. 7B, D), while there was no significant difference in the expression levels of p-IRF3 in the Poly (I:C)-treated WT mice and saline-treated WT mice in the cortex (Fig. 7A, C). The expression levels of p-IRF3 in the Poly (I:C)-treated AD mice were higher than those in saline-treated AD mice, and the results in the cortex and hippocampus were consistent (Fig. 7A, B, C, and D). The expression levels of p-NFκB were extremely low in saline-treated WT and Poly (I:C)-treated WT mice. The expression levels of p-NFκB in Poly (I:C)-treated AD mice were lower than those in saline-treated AD mice, with consistent results in both the cortex and hippocampus (Fig. 7E, F, G, and H).

Intraperitoneal Injection of Poly (I:C) Activates the TLR3/TRIF Pathway and Reduces the Inflammatory Burden in AD Mouse Brains

In this study, we examined the protein expression of p-IRF3 and p-NFκB in mouse brains using western blotting. The results revealed that the protein expression of p-IRF3 in Poly (I:C)-treated WT mice was higher than that in saline-treated WT mice. Moreover, in Poly (I:C)-treated AD mice, the expression levels of p-IRF3 were higher than those in saline-treated AD mice (Fig. 8A, B). Additionally, the expression of p-NFκB in the brains of saline-treated AD mice was higher than that in saline-treated WT mice. However, in Poly(I:C)-treated AD mice, the expression of p-NFκB was lower than that in saline-treated AD mice (Fig. 8C, D).

Fig. 8.

Poly(I:C) treatment activates the TLR3/TRIF pathway and reduces the inflammatory burden in AD mouse brains

After the behavioral experiment and MRI scan, the phosphorylation levels of IRF3 and NFκB were determined by western blotting. Transcript levels of inflammatory factors were measured by real-time qPCR. (A–B) Western blot representative bands and quantitative analysis of p-IRF3 and IRF3 in mouse brain tissue. (B) Western blot expression bands and quantitative analysis of p-NFκB and NFκB in mouse brain tissue. (E–H) Quantitative analysis of mRNA relative expression of IL-1β, TNF-α, IL-6, and IFN-β inflammatory factors in the brains of mice. Data are presented as mean ± SEM, n = 4/group. The statistical analysis included a one-way ANOVA and Tukey’s test. *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001 versus AD + Saline group.

Furthermore, we measured the mRNA expression levels of pro-inflammatory factors (IL-1β, TNF-α, and IL-6) and anti-inflammatory factors (IFN-β) in the brains of mice from each group using real-time quantitative PCR. The results revealed that the relative expressions of IL-1β, TNF-α, and IL-6 mRNA in the mouse brains of saline-treated AD mice were higher than those in saline-treated WT mice, However, the mRNA expression levels of IL-1β, TNF-α and IL-6 in Poly(I:C)-treated AD mice were significantly lower than those in saline-treated AD mice (Fig. 8E, F, and G). Additionally, the mRNA levels of IFN-β in the brains of Poly(I:C)-treated AD mice were significantly higher than those in saline-treated AD mice, while the mRNA levels of IFN-β in the brains of Poly(I:C)-treated WT mice were significantly higher than those in saline-treated WT mice (Fig. 8H). These results suggest that intraperitoneal Poly(I:C) injection mediates activation of the TLR3/TRIF pathway and reduces the transcription of pro-inflammatory factors while promoting the transcription of anti-inflammatory factors in the brains of AD mice.

Discussion

Polyinosinic-polycytidylic acid (poly(I:C)) is a synthetic analog of double-stranded RNA (dsRNA) and recognized by endosomal Toll-like receptor 3 (TLR3). Poly(I:C) is functionally validated for TLR3 potency and commonly used as a specific endosomal TLR3 agonist [29, 30]. In the present study, to investigate the role of activation of TLR3 in AD, the poly(I:C) was selected to treat the mice via intraperitoneally injection. After recognizing poly(I:C), TLR3 activates the transcription factor interferon regulatory factor 3 (IRF3), through the adapter protein Toll-IL-1 receptor (TIR) domain-containing adapter (TRIF or TICAM-1), leading to the production of type I IFNs [31]. The data of this study showed that systemic application of Poly(I:C) significantly increased p-IRF3 and IFN-β in brain tissue confirmed by immunofluorescence staining, Western blot, and qPCR analysis (Fig. 8), which strongly suggested that Poly(I:C) induced the activation of the TLR3 pathway.

In the present study, Poly (I:C) treated AD mice significantly reduced the time to find the platform compared to the no-treated AD mice (Fig. 1). In addition, Poly (I:C)-treated AD mice spent more time in the target quadrant than saline-treated AD mice (Fig. 1). This data demonstrated that intraperitoneal injection of Poly(I:C) improved cognitive function in AD mice. However, Poly (I:C) treatment did not affect the swimming. Incorporating additional parameters such as thigmotaxis and entropy would provide more comprehensive behavioral insights.

The brain-protective effect of Poly (I:C) is associated with the massive clearance of Aβ in the brain, the massive reduction of activated glial cells in white and gray matter, the decrease of the expression of pro-inflammatory factors, and the increase of the expression of anti-inflammatory factors. Our results show that pharmaceutical activation of the TLR3–TRIF pathway plays an important role in regulating the pathological progression of AD.

Scholtzova and colleagues demonstrated that intraperitoneal injection of TLR9 agonist class B CpG (cytosine–phosphate–guanine) oligodeoxynucleotides (ODNs) in AD mice improved their cognitive function [16]. Jean-Philippe and colleagues also demonstrated that repeated intraperitoneal injections of TLR4 receptor agonist monophosphoryl lipid (MPL) in AD mice significantly improved their cognitive function [15]. Our previous research indicated that the cognitive impairment of AD mice (APP⁺PS1⁺TLR2^−/−) was significantly impaired [14]. While the effects of targeting specific TLR receptors or applying TLR receptor agonists or inhibitors on cognitive function in AD mice differ, it is evident that TLR receptors play an essential role in the cognitive function of AD mice. In the current study, we administered the TLR3 receptor agonist Poly(I:C) systemically to AD mice and observed a significant improvement in their cognitive function.

The current consensus in the scientific community is that Aβ deposition, together with tau pathology and neuroinflammation, represents a fundamental pathological characteristic of AD. Although the precise pathophysiological mechanisms of AD are not fully understood, substantial evidence supports the pivotal role of Aβ in disease progression. Increased production or decreased clearance of Aβ are regarded as central events in AD [32]. Normally, Aβ_1–40 constitutes 90% of Aβ, with only small amounts of Aβ_1–42 and Aβ_1–43. However, in AD patients, the proportion of Aβ_1–42 in the brain rises [33]. Aβ_1–42 exhibits potent cytotoxicity and neurotoxicity, directly leading to the death of normal brain cells and neurons. The accumulation of Aβ_1–42 results in the formation of senile plaques, activating microglia and triggering inflammatory responses, leading to mitochondrial damage, disrupted energy metabolism, oxidative stress, and activation of apoptosis pathways [34].

Studies on TLR2 have shown that long-term inhibition of TLR2 reduces Aβ deposition and glial activation in brain tissue in AD mouse models [16]. Similarly, TLR9 agonists decrease Aβ deposition in brain tissue in AD mouse models [15], and TLR4 agonists reduce Aβ deposition and improve cognitive function in AD mouse models [14]. In our study, systemic administration of the TLR3 agonist Poly(I:C) every four days from 12 to 15 months of age significantly reduced Aβ deposition in the brains of AD mice.

Soluble Aβ oligomers are also currently the main toxicants associated with AD pathology. Hence, the measurement of Aβ oligomer levels in blood represents a simple and minimally invasive way to diagnose AD [35]. In this study, we measured Aβ_1–40 and Aβ_1–42 levels in mouse plasma. We found that the levels of Aβ_1–40 and Aβ_1–42 in mouse plasma decreased significantly after three months of application of the TLR3 agonist Poly(I:C).

Furthermore, white matter plays a crucial role in cognitive function [36]. Micro- and macrostructural abnormalities of white matter are common in AD patients, suggesting that white matter degeneration and demyelination may be important pathophysiological features in addition to the neuronal loss observed in the disease [27]. DTI has been widely used as a non-invasive clinical method to detect WM damage in the human brain. Previous studies have reported that FA values are an indicator of WM injury in DTI and decrease in conditions like traumatic brain injury (TBI), AD, and vascular dementia [32]. Our previous research demonstrated that TLR2 gene knockout exacerbates white matter damage in AD mice [14], and altered TLR4 signaling pathway activity is associated with cognitive deficits and white matter integrity in schizophrenia patients [33]. The results of this study showed that the systematic application of Poly(I:C) can delay the decrease of FA and DA values in the brains of AD mice.

In addition, we measured the expression of MBP using WB and immunofluorescence technology and found that the expression of MBP in Poly(I:C)-treated AD mice was significantly higher than that in saline-treated AD mice. Moreover, TEM ultrastructural imaging showed that the thickness of the myelin sheath of saline-treated AD mice was thicker than that of Poly(I:C)-treated AD mice. In summary, our results indicate that systemic application of Poly(I:C) reduces white matter damage in the brains of AD mice.

The loss of neurons and gray matter atrophy are the main pathological features of AD. The loss of neurons causes gray matter atrophy, ultimately leading to reduced cortical thickness and hippocampal volume [34]. Gray matter atrophy is closely related to the severity of cognitive impairment and is also a biomarker for early diagnosis of AD [28]. MRI is the preferred neuroimaging technique for diagnosing AD, as it allows for high tissue contrast and accurate measurement of the three-dimensional (3D) volume of brain structures, especially the size of the hippocampus and related regions [37]. The number of neurons in the brains of AD patients is significantly lower than that of normal people, especially in the CA1 and CA2 regions [38]. Previous research found that delayed administration of the TLR7 agonist gardiquimod (GDQ) after severe hypoxia–ischemia in the developing brain markedly ameliorated white and gray matter damage [38]. In the current study, the systemic application of Poly(I:C) was found to delay atrophy of the cortex and hippocampus in AD mice. Subsequent CV staining revealed a higher number of normal neurons in the cortical and CA1 areas in Poly(I:C)-treated AD mice compared to saline-treated AD mice. Thus, the systemic application of Poly(I:C) mitigates cortical and hippocampal atrophy as well as neuronal loss in AD mice.

Progressive memory impairment and cognitive decline in AD patients are closely linked to synaptic dysfunction and neuronal loss [39], and synaptic dysfunction is one of the prominent symptoms of AD [40]. LTP, a form of synaptic plasticity, is widely considered to underlie learning and memory [41]. Previous research by our group demonstrated significantly reduced LTP in the CA1 region of the hippocampus in TLR2 KO mice [42]. Animal experiments conducted by Patel et al. showed abnormal synaptic function in WT mice (TLR9 −/−) after TLR9 gene knockout [43]. Recent research has shown that Toll/TLRs have previously unsuspected functions related to development, cell fate regulation, cell number, neural circuit connectivity, and synaptogenesis [44]. The results of this study showed that intraperitoneal injection of Poly(I:C) significantly improved the synaptic function of AD mice.

An increasing body of evidence indicates that persistent immune inflammatory response is one of the core pathological features of AD. Neuroinflammation plays a positive role in the brain, such as promoting the removal of harmful substances from the brain and assisting in tissue repair. However, an uncontrolled and persistent inflammatory response can lead to a variety of chronic inflammatory diseases [45]. In the central nervous system (CNS), microglia and astrocytes are major sources of inflammation. Under pathological conditions, hyperactivated microglia, astrocytes, and released inflammatory factors create a neurotoxic environment, and sustained inflammatory response leads to neuronal loss and degeneration [46, 47]. The TLR3 agonist Poly(I:C) increases the expression of interferon β (IFN-β) and interferon regulatory factor 3 (IRF3) in the brain. In addition to innate antiviral immunity, IRF3 is also a key transcription factor regulating neuroinflammatory responses. IFN-β has also been shown to be crucial to the regulation of neuroinflammatory responses. Tarassishin et al. conducted a study involving the transduction of IRF3 into human primary microglia using a recombinant adenovirus, with two different immune stimuli. The results showed that IRF3 represses pro-inflammatory genes and enhances anti-inflammatory genes in microglia. Subsequently, Tarassishin et al. studied the transduction of IRF3 into human primary astrocytes using recombinant adenovirus, reporting similar results for astrocytes [48, 49]. Both microglia and astrocytes are the primary inflammatory cells in the brain. In the current study, the levels of protein markers for microglia and astrocytes in the brains of AD mice was significantly decreased after systemic application of poly(I:C). In addition, the expression of pro-inflammatory factors decreased, while the expression of anti-inflammatory factors increased.

MyD88 and TRIF are important adaptor proteins that mediate TLR signaling pathways [50]. Therefore, based on the difference of regulatory proteins, the TLR signaling pathway can be divided into the MyD88-dependent signaling pathway and the TRIF-dependent signaling pathway. TLR3 agonist Poly(I:C) activates the TRIF-dependent signaling pathway, which recruits its downstream protein kinases, leading to the activation of IRF3 and IRF7 and regulating the expression of type I interferon (IFN). In addition, TRIF can phosphorylate IKK through receptor-interacting protein kinase 1 (RIP1)-mediated activation of tumor necrosis factor receptor-associated factor 6 (TRAF6), activate NF-kB, and regulate the transcription of inflammatory cytokines [10, 51]. However, according to Zhao et al., Poly(I:C) application upregulates the expression of IRF3, and IRF3 inhibits the TRIF-mediated NF-κB signaling pathway [52]. In the present study, we found that p-IRF3 and p-NF-κB were most expressed in astrocytes in AD mice. The expression levels of p-IRF3 in Poly(I:C)-treated AD mice were higher than those in saline-treated AD mice, while the expression levels of p-NF-κB in Poly(I:C)-treated AD mice were lower than those in saline-treated AD mice. The protective mechanism of systemic application of Poly(I:C) in AD may be related to the activation of the TLR3-TRIF pathway in astrocytes, which increased the phosphorylation of IRF-3 in the astrocytes and the transcription level of IFN-β in the brain. The increased IFN-β upregulates the expression of anti-inflammatory factors. In addition, increased IFN-β inhibits the phosphorylation of NF-κB in astrocytes, and leading to the decrease of pro-inflammatory factors, including IL-1β, IL-6, and TNF-α.

Poly(I:C) is a compound that mimics a transient viral infection, and its immediate pharmacological effects may diminish within a few days. However, the downstream effects of TLR3 activation, such as the changes in gene expression, regulation of neuroinflammation, and modulation of synaptic plasticity, are likely to persist beyond the immediate timeframe of Poly(I:C) clearance. Previous study reported that Poly(I:C) treatment induces a robust and sustained activation of the TLR3 signaling and that administration of Poly(I:C) every 4 days for 36 days in mice elevated and maintained the levels of TLR3 signaling components, such as TLR3 and IRF3 [53]. Nevertheless, the precise pharmacokinetics of Poly(I:C) and its long-term impact on the brain microenvironment, particularly in the context of AD deserve further study. Moreover, the present study detected the mRNAs of the IL-1β, IL-6, TNF-α, and IFN-β, the levels of these proteins would help the comprehensive understanding of the inflammatory response.

Poly(I:C), as a synthetic analog of dsRNA, is recognized by endosomal TLR3. After recognizing poly(I:C), TLR3 activates the transcription factor interferon regulatory factor 3 (IRF3) and IFN-β as well as NF-κB. The results from the co-staining demonstrated that the p-IRF3 and p-NF-κB were predominantly observed in astrocytes rather than in microglia. The reason for this phenomenon may be that astrocytes are more involved in the later stages of AD pathology than microglia. Ideally, additional approaches such as cell-specific knockout models or nuclear IRF3 localization would provide more direct evidence. Furthermore, their responses to poly(I:C) may also differ. The exact roles of astrocytes and microglia in AD and their different responses to Poly(I:C) treatment require further investigation.

Previous studies demonstrated that in APP/PS1 mice, amyloid-β (Aβ) plaques were detected starting at 6 months of age, and cognitive deficits were observed as early as 9 months of age [54–56]. Clinically, most patients seek medical intervention only after experiencing significant symptoms, such as cognitive impairment, emotional disturbances, and other well-defined clinical manifestations. These symptoms typically indicate the later stages of AD. Understanding and addressing disease progression at this late stage is critical to translating research findings into clinical practice. In this study, to simulate the late-stage AD scenario, we specifically used late-stage AD mice as an experimental model, in which treatment is usually initiated after significant disease progression. Our data suggested that systemic administration of poly(I:C) could alleviate rather than restore neurobehavioral deficits and exacerbated pathology in the late-stage AD mice.

In summary, our study demonstrates that the systemic application of TLR3 agonist Poly(I:C) attenuated the damage to white matter and gray matter in AD brain, improved the cognitive function, and reduced the deposition of Aβ_1–42 in the brain and the content of Aβ_1–40 and Aβ_1–42 in peripheral blood. The underlying mechanism may be related to the up-regulation of p-IRF3 that increases the expression of anti-inflammatory factors and the inhibition p-NF-κB that reduces the expression of pro-inflammatory factors. In this study, to minimize potential interference caused by hormonal fluctuations in female mice, we selected only male mice. Given the significant role that gender differences play in AD, future studies should include both male and female mice to provide a more comprehensive understanding of the gender-related differences in the observed effects.

Supplementary Information

Below is the link to the electronic supplementary material. 

Author Contribution

FH, TZ, and CZ contributed to the concept and design of the study, acquisition and analysis of data, and drafting and revising the manuscript. HZ, FS, WN, and HG contributed to the concept and design of the study and analysis of data. XJ, SW, DW, FD, ZS, GJ, JH, and YZ contributed to the acquisition of data. JZ, XY, and HS contributed to revising the manuscript. All authors have reviewed and approved the final version of the manuscript.

Funding

This work was supported by the National Nature Science Foundation of China (82171420).

Data Availability

No datasets were generated or analysed during the current study.

Declarations

Ethical Approval

This study was performed in line with the principles of the Declaration of Helsinki. Approval was granted by the Ethics Committee of Xuzhou University (08/25/2023/No. 202309T0).

Consent to Participate

The authors confirm that the consent to participate of a human subject was not applicable in the present study.

Consent for Publication

The authors confirm that the consent for publication of a human subject was not applicable in the present study.

Competing interests

The authors declare no competing interests.