Secoisolariciresinol diglucoside ameliorates Alzheimer-like lesions by increasing MKP-1

Abstract

Background

Secoisolariciresinol diglucoside (SDG), a phytoestrogen, has been demonstrated to exert anti-inflammatory and neuroprotective effects. Mitogen-activated protein kinase (MAPK) phosphatase-1 (MKP-1) serves as a critical negative regulator of MAPK signaling pathways, and the MAPK signaling pathways play a significant role in the pathogenesis of Alzheimer's disease (AD). However, it remains unclear whether SDG ameliorates Alzheimer-like lesions by regulating the MAPK pathway through increasing MKP-1.

Objective

We aimed to investigate the impact of SDG on the Alzheimer-like lesions of AD mice and its mechanisms.

Methods

Three-month-old 5×FAD mice were treated with SDG (50 mg·kg-1·d-1, i.g.) for 2 months. Learning and spatial memory function was assessed with the behavioral test. Immunofluorescence and Thioflavine-S staining was assessed with the levels of amyloid-β (Aβ) plaques in the cortex and hippocampus. Western blot was performed to evaluate the level of learning memory-related proteins, hyperphosphorylated tau, APP-related proteins, and MAPK phosphorylation. Besides, knockdown of MKP-1 in N2A/APP cells to investigate whether SDG regulates the MAPK signaling pathway by increasing MKP-1.

Results

We found that SDG significantly enhanced learning and spatial memory while recovering PSD95, PKA-Cα, and synaptophysin levels in 5×FAD mice. SDG reduced Aβ plaques, tau phosphorylation at Ser 199/214/262/396 and Thr 231, alleviated the phosphorylation of MAPKs (JNK, ERK1/2, P38), and increased p-GSK-3β (Ser9), while decreased activation of microglia (Iba-1) and astrocytes (GFAP). Moreover, knockdown of MKP-1 in N2A/APP cells inhibited the regulatory effect of SDG on APP, ERK1/2 and JNK.

Conclusions

SDG ameliorates Alzheimer-like lesions may be related with increasing MKP-1.

Keywords

Alzheimer’s disease amyloid-β MKP-1 secoisolariciresinol diglucoside tau

Introduction

Alzheimer's disease (AD), also referred to as senile dementia, is among the most prevalent age-related neurodegenerative disorders affecting the central nervous system. The disease is characterized by an insidious onset, with primary neuropsychiatric manifestations including progressive memory decline, marked cognitive deficits, significant language impairments, and pronounced alterations in personality and behavioral independence.^1,2 The severity of these symptoms is positively correlated with advancing age, often culminating in profound dementia, widespread cognitive decline, and ultimately death due to severe complications.³ According to reports, the global number of people living with dementia is projected to rise to 153 million by 2050.⁴ The growing burden on patients has evolved into a major public health challenge, placing significant economic strain on societies, underscoring the urgent need for the development of novel therapeutic strategies.^5,6

Amyloid-β (Aβ) plaques and neurofibrillary tangles (hyperphosphorylated tau) are the two hallmark neuropathological features of AD.⁷ Current therapeutic strategies for AD largely focus on reducing Aβ production or inhibiting tau hyperphosphorylation.⁸ However, due to the insidious onset of AD, most patients are already in the middle to late stages of the disease by the time they seek medical attention, making it challenging to achieve satisfactory outcomes with single-target drugs directed at either Aβ or tau.⁹

Emerging evidence suggests that neuroinflammation plays a critical role in the early stages of AD. This neuroinflammatory response is predominantly mediated by glial cells, including microglia and astrocytes.⁹ Recently, the anti-neuroinflammatory drug GV-971 (glycosylated GV-971), which targets multiple pathways in AD by modulating gut microbiota, has demonstrated some clinical efficacy. However, its mechanism of action remains controversial and requires further investigation.¹⁰

Clinical studies have shown that menopausal estrogen replacement therapy is associated with a reduced risk of AD, likely due to the neuroprotective effects of estrogen.¹¹ Estrogen has been shown to exert neuroprotective effects by inhibiting microglia-mediated neuroinflammation and suppressing astrocyte activation.¹² Nevertheless, in clinical trials, exogenous estrogen use in perimenopausal women has been linked to an increased risk of breast and endometrial cancers.¹³ Interestingly, studies indicate that phytoestrogens, plant-derived compounds with estrogen-like activity, may provide similar neuroprotective benefits while circumventing the cancer risks associated with exogenous estrogen therapy in perimenopausal women.¹⁴

Phytoestrogens share a structural similarity with 17β-estradiol (E2), allowing them to readily bind to estrogen receptors and mediate estrogenic responses.¹⁵ In animal models of AD, phytoestrogens have demonstrated antioxidant, anti-apoptotic, and neuroprotective effects.¹⁶ They have also been shown to improve learning and memory functions while reducing Aβ production and tau protein hyperphosphorylation.^17,18 Moreover, clinical trials have indicated that supplementation with specific levels of soy isoflavones can effectively enhance spatial and learning memory in both men and postmenopausal women.¹⁹ These findings suggest that phytoestrogens may exert beneficial effects on both the clinical symptoms and neuropathological features of AD.

Flax lignans (FL), also known as secoisolariciresinol diglucoside (SDG), are a class of naturally active phytoestrogens structurally similar to human estrogens, produced through the polymerization of phenylpropanoid bilayers. Flaxseed is the richest natural source of SDG, with its content being 75∼800 times higher than that found in other oilseeds, cereals, pulses, fruits, and vegetables.²⁰ Due to its anti-inflammatory, antioxidant, hypolipidemic, and neuroprotective properties, SDG has shown therapeutic potential for the treatment of cardiovascular, diabetic, atherosclerotic, and urological diseases.²¹

Preclinical studies have demonstrated that SDG is effective in various contexts; for instance, SDG has been shown to alleviate aseptic encephalitis by improving blood-brain barrier permeability and reducing systemic inflammation.²² These findings suggest that SDG represents a promising and versatile candidate for the treatment of neurodegenerative diseases. In this study, we sought to further explore the biological functions of SDG by utilizing animal models and cellular models of AD to investigate its potential to improve learning and memory as well as mitigate pathological changes associated with AD.

Methods

Reagents and antibodies

SDG was purchased from Chengdu Phytolabeling & Chemical Pure Co (Cat#: PCS0939, Chengdu, China). The main antibodies used in this study are listed in Table 1. The enhanced chemiluminescence (ECL) kit was obtained from Thermo Scientific (Cat#: 1863096, Rockford, IL, USA).

Table 1.

Primary antibodies used in this study.

Antibody	Type	Dilution; apply	Source/reference	Cat. no.
β-tubulin	pAb	1:8000; WB	Bioword Technology	AP0064
4G8	mAb	1:400; IF	Abcam	ab77800
APP(pT668)	pAb	1:2000; WB	Abcam	ab194744
APP(Y188)	mAb	1:20000; WB	Abcam	ab32136
BACE1	mAb	1:1000; WB	Abcam	ab183612
ERK	mAb	1:1000; WB	CST	4695S
GFAP	mAb	1:200; IF	CST	3670S
GSK3β	mAb	1:1000; WB	CST	9832S
JNK	pAb	1:1000; WB	CST	9252S
Iba1	mAb	1:200; IF	GeneTex	GT10312
MKP-1 (E-6)	pAb	1:300; WB	SANTA CRUZ	sc-373841
NLRP3	pAb	1:1000; WB	SAB	29125
PSD95	pAb	1:1000; WB	Servicebio	GB11277
PKA-Cα	pAb	1:1000; WB	CST	4782S
P-JNK	mAb	1:1000; WB	CST	4668S
P-ERK1/2	mAb	1:1000; WB	CST	4370S
P-tau (Ser 199)	mAb	1:5000; WB	Abcam	ab81268
P-tau (Ser 214)	pAb	1:1000; WB	Invitorgen	2141602
P-tau (Thr 231)	pAb	1:1000; WB	Invitorgen	PA5-117230
P-tau (Ser 262)	pAb	1:1000; WB	Abcam	ab131354
P-tau (Ser 396)	mAb	1:10000; WB	Abcam	ab109390
P-P38	pAb	1:1000; WB	CST	9211S
P38	mAb	1:1000; WB	SAB	48644-2
P-GSK3β (Ser 9)	mAb	1:1000; WB	CST	5558S
P-PP2A (Thy 307)	pAb	1:1000; WB	Invitorgen	PA5-36874
PP2Ac	pAb	1:1000; WB	CST	2038S
Synaptophysin	mAb	1:8000; WB	Servicebio	GB12553
Tau-5	mAb	1:1000; WB	Abcam	ab80579

WB: western blotting; IF: immunofluorescence; mAb: monoclonal antibody; pAb: polyclonal antibody.

Animals and cells

The C57BL/6J mice (Wide type, WT) used in the experiment were purchased from the Guangdong Medical Laboratory Animal Center (Guangzhou, China). The AD model mice are 5×FAD transgenic mice were gifted by Xifei Yang, researcher of Shenzhen Disease Control and Research Center. 5×FAD mutations include five familial mutant genes, including overexpression of 3 mutant human APP-related genes (Swedish K670N/M671L, Florida I716 V; London V717I) and 2 Presenilin 1 (PSEN1)-associated mutant genes (M146L and L286 V). 5×FAD male mice were combined with C57BL/6J wild-type female mice for caged breeding offspring. litters were weaned at postnatal day 21 and group-housed by sex. Subsequently, they were either assigned to experiments or retained for breeding. Co-littered wild-type mice (WT mice) as a control group. Only male mice were used in the experiments to minimize the effects of hormones in behavioral analysis. All mice were housed under specific pathogen-free conditions under controlled temperature (21–25°C) with an alternating 12-h light/12-h dark cycle and standard laboratory diet and water at Guangzhou Medical University. All animal experiments were performed in accordance with protocols approved by the Institutional Animal Care and Use Committee of Guangzhou Medical University.

Mouse neuroblastoma Neuro2a (N2A) cells /WT and cells stably expressing mutant APP (N2A/APP) were purchased from Fenghui Biotechnology (Cat#: CL0383 for N2A/WT, CL0243 for N2A/APP; Changsha, China). The cells were cultured in Dulbecco’s modified Eagle’s medium (Gibco, NY, USA) containing 10% fetal bovine serum, 0.5% 100 U/mL penicillin, and 100 μg/mL streptomycin in a 5% CO2 incubator at 37°C.

Drug administration

Mice were divided into four groups; saline-treated WT mice (WT + NS); SDG-treated WT mice (WT + SDG); saline-treated 5×FAD mice (5×FAD + NS); SDG-treated: 5×FAD mice (5×FAD + SDG). According to the instructions, SDG was prepared into a 50 mg/kg suspension with 0.9% saline before application. To investigate the effect of SDG on the learning and memory abilities of 5×FAD mice, WT or 5×FAD male mice were treated with SDG (50 mg·kg⁻¹·d⁻¹, i.g.) or vehicle (0.9% saline) for 2 months. During this period, the weight of the mice and their intake of food and water were recorded daily.

Cells are treated with different concentrations of SDG (1, 5, 10, 20 μM) for 24 h.

Morris water maze test

To evaluate the effects of SDG on spatial memory, the Morris water maze test was performed as previously described with minor modifications.²³ The swim speed (cm/s), time ratio of the first to the third quadrant, swim distance (mm), escape latency (s) in initial training, latency to enter the target (platform location area), platform-crossing times, and platform crossover number were recorded through an automated tracking system (Smart video tracking system, Panlab; Harvard Apparatus).

Y-maze test

Short-term spatial memory was evaluated using the Y maze test. In this test, the mice were placed in the center of the Y-maze and allowed to freely shuttle through three arms within 5 min. The sequence and total number of mice entering and exiting each arm was recorded for each mouse. After the test of each mouse, clean the feces and wipe the inside of the Y maze with 75% alcohol until the alcohol evaporates completely before continuing with the next mouse test to prevent the odor from affecting the shuttle behavior of subsequent mice. Spontaneous alternations were recorded by using a camera and manually counted.

Novel object recognition test

Novel object recognition (NOR) test was used to evaluate recognition memory in mice.²⁴ The NOR test proceeded in an open field device with a side length of 60 cm and a height of 30 cm. Before the training begins, each mouse was placed in the test box to adapt for 10 min to adapt the environment. During the training phase, the mouse was allowed to freely explore for 8 min in the open field containing two identical objects (yellow cubes, A and B) placed the same distance from the walls. The number and time of mouse contacts with objects A and B was recorded. After the training, the mice return back to its mouse cage, and then the open field and objects were cleaned with 75% alcohol and air-dried before the next experiment. After all animals have completed the familiarization process, two familiar objects are replaced, one with a familiar yellow cube (B) and the other with a novel object (red cylinder, C). In the present experiments, two objects (B and C) were placed in the same position as before and the animals were allowed to freely explore for 8 min. The detection time of familiar and new objects in mice was recorded for analysis.

Brain tissue preparation and immunofluorescence

After the behavior test, mice were anesthetized by 1% inhaled isoflurane and then perfused with phosphate-buffered saline (PBS) followed by 4% paraformaldehyde. Then, the brain tissues were immediately removed from the skull and postfixed in 4% paraformaldehyde overnight at 4°C for 24 h followed by 30% sucrose-solution for two days at 4°C. As previously described, coronal serial sections were cut at 20 mm thickness with a vibratome (VT1000S, Leica, Nussloch, Germany). The slices were permeabilized with PBS containing with 0.2% Triton-X and blocked with 5% BSA for 1 h at room temperature followed by incubation overnight at 4°C with primary antibody (see Table 1) diluted in blocking solution. Then the sections were again washed and incubated with Alexa 488-conjugated goat anti-rabbit IgG (1:200 dilution), Alexa 488-conjugated goat anti-mouse IgG (1:200 dilution), and Alexa 549-conjugated goat anti-mouse IgG (1:200 dilution) were added to the slices and incubated 2 h in dark, washed three times with PBS for 15 min, and sections were mounted and incubated with DAPI (CST, Danvers, MA, USA), then were coverslipped. Immunofluorescence was visualized and captured on a laser scanning confocal microscope (Alsi, Nikon, Japan). All images were analyzed using ImageJ software (NIH, Bethesda, USA).

Thioflavine S staining

The Aβ plaques in cortex and hippocampus were detected by thioflavine S staining (ThS). After a wash with PBS for 15 min, the slices were stained with freshly prepared 0.3% ThS solution for 8 min at room temperature in dark and then washed twice in 50% ethanol solutions for 5 min each time. Subsequently, the brain tissue was washed in PBS for 5 min and coverslipped, then viewed by a confocal laser canning microscope (A1si, Nikon, Japan).

Western blot

For biochemical analysis, the cortex and hippocampus were separated from brain tissue and stored at −80°C until use. Before the experiment, the frozen tissue of the mice brain were homogenized in RIPA buffer (Cat#: FD008, Fude Bio, Hangzhou, China) and then the protein concentration of the sample was determined using the BCA kit (Cat#: 89880, Pierce, MA, USA). After preparing the protein into the same concentration sample, separated the same accounts of protein (30 μg) with different concentrations of sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE, 8%, 10% or 12%) at 80 V for 30 min, 100 V for 90 min, and then transferred onto the polyvinylidene difluoride membranes. Then the membrane were blocked at room temperature with PBS solution containing 5% non-fat milk for 1 h, and incubated it overnight with specific primary antibody (see Table 1) at 4°C. After washing with PBST twice for 10 min, we then incubated the membranes with secondary antibody conjugated to horseradish peroxidase (HRP) for 1 h at temperature and detection was realized using Pierce ECL kit (Thermo Scientific, Waltham, MA, USA), visualized with the Gel Doc XR System (Bio-Rad). Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and β-tubulin levels were analyzed as a loading control, and the membranes were analyzed using Image J (NIH, Bethesda, USA). The specific steps of western blotting in cell experiments are the same as above.

RNA interference

RIBOBIO (Guangzhou, China) is responsible for the production and synthesis of MKP-1 specific siRNA. The siRNA sequences are depicted below.

siRNA targeting MKP-1; siMKP-1: 5′-GCATCACCGCCTTGATCAA-3′;

Cells were transfected with siRNA using Lipofectamine RNAiMAX (Invitrogen, Carlsbad, CA, USA) reagent according to the manufacturer's guidelines. 24-h post-transfection, cells were treated with SDG (10 μM). Following a 24-h period, the cells were harvested and a total protein extraction was conducted for the subsequent western blot procedure.

Statistical analysis

Statistical were analyzed using GraphPad Prism 8.0 (GraphPad Software, Inc., La Jolla, CA, USA). All data were expressed as the mean ± SEM. Statistical significant between groups were assessed by one-way ANOVA or Student's t-test. p < 0.05 were considered statistically significant.

Results

SDG improve learning memory in 5×FAD mice

Initially, the Y-maze spontaneous alternation test and the NOR test were carried out. Results revealed that 5×FAD mice exhibited fewer sequential entries into the three different arms of the Y-maze compared to WT + NS mice, indicating significant short-term memory deficits in the 5×FAD model group (Figure 1A). Similarly, the NOR test confirmed impairments in short-term memory in the 5×FAD mice (Figure 1B). These two behavioral experiments collectively demonstrated that 5×FAD mice had substantially reduced spatial memory and object recognition abilities. However, 5×FAD mice treated with SDG showed significant improvements in both spatial memory and object recognition, suggesting that SDG has the potential to partially ameliorate short-term spatial and object memory deficits in the 5×FAD mouse model.

In the Morris water maze experiment, spatial learning and memory abilities were assessed by measuring the escape latency to find the hidden platform, with trials conducted four times per day. No significant differences in swimming speed were observed between the groups (Figure 1C), indicating that all mice had normal locomotor abilities.

With normal motor function confirmed, we evaluated spatial learning ability starting from the third day of training. SDG-treated 5×FAD mice exhibited significantly shorter escape latencies compared to untreated 5×FAD mice (Figure 1D), suggesting that while spatial learning was impaired in 5×FAD mice, SDG treatment partially alleviated these deficits.

To assess spatial memory, on the sixth day of the experiment, the hidden platform was removed, and the time spent in the target quadrant, the number of platform crossings, and the swimming trajectory were recorded during a 60-s trial. Untreated 5×FAD mice demonstrated significant impairments in spatial memory, including longer latency to enter the target quadrant for the first time (Figure 1E), a lower ratio of time spent in the target quadrant compared to the first three quadrants (Figure 1F), shorter time spent in the platform area (Figure 1G), fewer platform crossings (Figure 1H), and reduced swimming distance within the target quadrant (Figure 1I). In contrast, SDG-treated 5×FAD mice showed significant improvements across all these metrics (Figure 1E–1). Representative swimming trajectories from the water maze are displayed in Figure 1J.

In summary, the Morris water maze results demonstrated that SDG treatment significantly improved the spatial learning abilities of 5×FAD mice and alleviated their spatial memory impairments.

SDG increase the expression of learning memory-related proteins in the brain of 5×FAD mice

Based on the findings of previous behavioral experiments, which demonstrated that SDG improved learning and memory functions in 5×FAD mice, we further investigated the levels of proteins associated with learning and memory. Our results showed that the expression levels of PSD95, PKA-Cα, and synaptophysin (SYP) were significantly reduced in 5×FAD model mice compared to the control group. However, treatment with SDG restored the expression of these proteins to varying degrees (Figure 2A, E). These findings suggest that SDG may improve learning and memory by regulating the expression of key proteins involved in synaptic function and plasticity.

SDG reduce Aβ plaque deposition in the brain of 5×FAD mice

In this study, we first employed ThS staining to assess amyloid plaque deposition. The results revealed a significant increase in amyloid plaques in the cortical and hippocampal regions of the brains of 5×FAD mice. In contrast, SDG treatment markedly reduced the number of plaques in these regions (Figure 3A).

To further confirm these findings, we utilized the 4G8 antibody, which specifically recognizes Aβ/APP, in conjunction with immunofluorescence staining. This analysis showed a significant reduction in the distribution and content of Aβ plaques in the brains of SDG-treated 5×FAD mice compared to untreated 5×FAD mice (Figure 3D).

Taken together, these results indicate that SDG significantly reduces Aβ levels and the number of amyloid plaques in the cortical and hippocampal regions of 5×FAD mouse brains.

SDG reduce hyperphosphorylated tau and APP-related proteins expression in the brain of 5×FAD mice

To investigate whether SDG affects tau phosphorylation in the cerebral cortex of 5×FAD mice, we examined the levels of phosphorylated tau in both the cortex and hippocampus of each mouse. Phosphorylated tau at specific sites (Ser 199/214/262/396 and Thr 231) were measured using site-specific antibodies (Figure 4A, C). The results showed no significant differences in total tau levels between the groups, as detected with the tau-5 antibody.

In the cortical region, phosphorylated levels of tau were significantly elevated at the Ser 214/262/396 and Thr 231 sites in 5×FAD model group compared to control group. SDG treatment significantly reduced phosphorylation levels at Ser 214/262/396 and Thr 231 in 5×FAD mice (Figure 4B). Similarly, In the hippocampal region, phosphorylated levels of tau were significantly elevated at the Ser 199/214/396 and Thr 231 sites in 5×FAD model group compared to control group. SDG treatment significantly reduced phosphorylation levels at Ser 199/214/396 and Thr 231 in 5×FAD mice (Figure 4D).

Our results revealed that the expression levels of APP(pT668), APP(Y188), and BACE1 were significantly elevated in the 5×FAD model group compared to the control group. Notably, SDG treatment markedly reduced the expression levels of these APP-related proteins compared to the untreated 5×FAD group. This suggests that SDG inhibited the abnormal activation of BACE1 and decreased the expression of both APP and BACE1, thereby reducing Aβ production (Figure 4E, G). Furthermore, the levels of soluble amyloid precursor protein were significantly decreased in both SDG-treated groups compared to the 5×FAD model group, with levels approaching those observed in WT mice. These findings indicate that SDG may reduce Aβ production in the brain by mitigating the abnormal metabolism of APP.

Effect of SDG on the upstream kinase and esterase of tau protein in 5×FAD mice

Because SDG reduced phosphorylated tau in 5-month-old 5×FAD mice, we further investigated whether SDG affects the levels of kinases involved in tau phosphorylation, including extracellular signal-regulated kinase 1/2 (ERK1/2), c-Jun N-terminal kinase (JNK), p38, glycogen synthase-3β (GSK-3β), and protein phosphatase 2A (PP2A).²⁵ Phosphorylation levels of JNK, ERK1/2, and P38 were significantly increased in 5×FAD mice compared to control mice. Interestingly, oral administration of SDG effectively attenuated the abnormal phosphorylation of JNK, ERK1/2, and P38 in the cortex and hippocampus of 5×FAD mice. Although the phosphorylated P38 levels in the cortex of SDG-treated 5×FAD mice were reduced compared to untreated 5×FAD mice, this reduction did not reach statistical significance.

These findings suggest that oral administration of SDG mitigates the abnormal phosphorylation of JNK, ERK1/2, and P38 in the brain tissues of 5×FAD mice (Figure 5A, C), providing further insight into the molecular mechanisms of SDG's potential anti-AD effects.

To further investigate, we assessed the expression levels of GSK3β and PP2A in the cortex and hippocampus by examining P-GSK3β (Ser9)/GSK3β and P-PP2A (Tyr307). In the 5×FAD model group, P-GSK3β (Ser9)/GSK3β levels were significantly decreased, indicating elevated GSK3β activity, while P-PP2A (Tyr307) levels were significantly increased, reflecting reduced PP2A activity. Notably, in 5×FAD mice treated with SDG, GSK3β activity was suppressed, as evidenced by increased P-GSK3β (Ser9)/GSK3β levels, and PP2A activity was enhanced, indicated by decreased P-PP2A (Tyr307) levels.

These findings suggest that SDG mitigates the abnormal phosphorylation of tau protein in 5×FAD mice by reducing the activity of upstream kinases such as GSK3β and enhancing the activity of upstream phosphatases like PP2A (Figure 5).

Secoisolariciresinol diglucoside inhibit the activation of neuroglia in the brain of 5×FAD mice

We performed immunofluorescence staining with antibodies against Iba-1 and GFAP (Figure 6A, G). Compared with the control group, in the Iba-1 immunofluorescence staining results, we found that mice in the 5×FAD model group had enhanced positive Iba-1 fluorescence staining in CA1, CA3, DG, and the cortex, and significantly increased microglia number and volume compared with the control group, whereas 5×FAD mice given SDG had significantly weaker positive Iba-1 staining in the corresponding regions and microglia The Iba-1 positive staining was significantly reduced in the corresponding areas of 5×FAD mice given SDG, and the volume and number of microglia were significantly reduced (Figure 6A), suggesting that SDG could attenuate the activation of microglia. At the same time, the 5×FAD model mice showed increased positive staining for GFAP in CA1, CA3, DG and the cortex, with an increase in the number of astrocytes and a significant increase in cytosolic volume. In contrast, the GFAP positive staining in the corresponding areas of 5×FAD mice given SDG gavage was significantly weaker, and the number and morphology of the cytosol were significantly reduced (Figure 6G), suggesting that SDG was also able to reduce the activation of astrocytes in the AD model group; these results suggest that SDG may reduce the neuroinflammatory response by inhibiting the activation of microglia and astrocytes.

Molecular mechanisms of secoisolariciresinol diglucoside amelioration of Alzheimer-like lesions

Mitogen-activated protein kinases (MAPKs), including ERK1/2, JNK and p38, play a pivotal role in cellular signal transduction and significantly influence the pathogenesis and progression of AD.^26–28 Furthermore, mitogen-activated protein kinase phosphatase-1 (MKP-1) is a key negative regulator of MAPKs.²⁹ To further investigate the role of MKP-1 in AD, we examined its expression in the brains of AD mice. The results showed that MKP-1 levels in the cortex and hippocampus were significantly decreased in 5×FAD mice. However, administration of SDG significantly increased MKP-1 levels in these regions of 5×FAD mice (Figure 7A, C).

The N2A/APP cell line is a well-established AD model that overexpresses APP and simultaneously increases the phosphorylated tau (Figure 7E–G). Therefore, N2A/APP was used to examine the effects of SDG on APP, tau, and MAPKs (ERK1/2 and JNK). Treatment with 10 μM and 20 μM SDG significantly reduced the expression levels of APP (Y188), phosphorylated tau (pS396), and the abnormal phosphorylation of JNK and ERK1/2 in N2A/APP cells (Figure 7H–L). Additionally, SDG treatment markedly increased MKP-1 expression (Figure 7M).

To further confirm the critical role of MKP-1 in the neuroprotective effects of SDG, N2A/APP cells were transfected with siRNA to knock down MKP-1. The results demonstrated that the SDG-induced reduction in APP (Y188), ERK1/2 and JNK expression was significantly inhibited following MKP-1 knockdown, compared with the corresponding controls (Figure 7N).

These findings suggest that SDG reduces Aβ production by upregulating MKP-1 expression and alleviates tau pathology by regulating MAPK signaling pathways.

Discussion

AD is an irreversible neurodegenerative disease often accompanied by dementia. Many theoretical theories have been proposed for the pathogenesis of AD, among which the more popular ones are the Aβ deposition theory, the neuroinflammation theory, and the theory of abnormal phosphorylation of tau proteins.³⁰

Estrogen, as a class of neuroactive steroid hormones, has been demonstrated to exert significant neuroprotective effects in various neurological disorders.^11,31 Based on this property, estrogen replacement therapy has demonstrated potential therapeutic value in AD.³² However, concerns over its adverse effects—including increased risks of thrombosis, stroke, and myocardial infarction—have limited its clinical translation.³³ Against this backdrop, phytoestrogens, structurally similar to estrogen yet offering superior safety profiles, have emerged as highly promising alternative strategies.

SDG is a phytoestrogen derived from flaxseed, exhibiting multiple biological activities including anti-inflammatory, antioxidant, lipid-regulating, and neuroprotective effects.²² Research has confirmed that SDG demonstrates beneficial effects on cardiovascular disease, diabetes, atherosclerosis, and urological disorders.²¹ Furthermore, SDG can reduce central nervous system inflammatory damage by regulating blood-brain barrier permeability, thereby limiting the infiltration of peripheral inflammatory factors into the central nervous system.³⁴ SDG alleviates radicular pain in a rat model of painful radiculopathy by suppressing astrocyte activation and reducing the accumulation of reactive oxygen species and reactive nitrogen species.³⁵ Interestingly, our study found that SDG suppresses the activation of astrocytes and microglia in the brains of 5×FAD mice, potentially through increased MKP-1. MKP-1 has been reported to be expressed in glial cells and participates in the regulation of neuroinflammation.^36–38 MKP-1 and PP2A interact to regulate microglial activation in a rat model of Parkinson's disease.³⁹ MKP-1 is a key negative regulator of MAPKs.²⁹ Overexpression of MKP-1 in BV-2 microglia cells blocked lipopolysaccharide-induced p38 and JNK phosphorylation while also suppressing the release of proinflammatory mediators and reactive oxygen species.^40,41 Furthermore, PPAR-α agonists upregulate MKP-1 mRNA levels to inhibit CCL2/MCP-1 protein transcription and secretion, thereby suppressing IFN-γ-stimulated JNK phosphorylation in astrocytes.⁴² However, the mechanism by which SDG inhibits glial cell activation requires further investigation.

Aβ deposition in the cerebral cortex is a key pathological hallmark of AD. The deposition of Aβ plaques leads to the death of neurons and induces impairment of learning and memory functions.³⁰ Previous studies have demonstrated that amyloid plaques begin to form in 5×FAD mice at approximately 2 months of age and accumulate to significantly higher levels between 4 and 5 months of age.⁴³ According to reports, SDG ultimately inhibits brain Aβ deposition in APP/PS1 mice by promoting the production of gut microbial metabolites.⁴⁴ Similarly, we provide the first evidence that oral administration of SDG (50 mg·kg⁻¹·d⁻¹) significantly reduces Aβ deposition in 5-month-old 5×FAD female mice, an effect potentially linked to modulation of amyloidogenic processing. Aβ is derived from APP, and SDG treatment lowers APP levels in both the cortex and hippocampus of 5×FAD mice. Aβ formation requires sequential APP cleavage by β-secretases and γ-secretases.⁴⁵ BACE1 (β-site APP cleaving enzyme 1) acts as the rate-limiting enzyme that catalyses the first step in Aβ production.⁴⁶ We found that BACE1 is markedly elevated in 5×FAD mice, whereas SDG treatment markedly attenuates this elevation. Research indicates that the phosphorylation level of Thr668 (T668) in the cytoplasmic domain of APP is significantly elevated in both AD patients and mouse models.^47–49 APP(pT668) may promote APP cleavage by BACE1 and Aβ peptide production,^47,50 and APP has been reported to be phosphorylated by multiple protein kinases (including GSK3β and JNK).⁵¹ Therefore, we further investigated the effects of SDG on protein kinases. We found that SDG inhibited GSK3β and JNK activity, reducing APP and APP(pT668). Furthermore, siRNA-mediated knockdown of MKP-1 in N2A/APP cells blocked SDG's ability to decrease p-JNK and APP. Collectively, these data indicate that SDG may reduce phosphorylated APP by regulating GSK3β and the MKP-1/JNK signaling pathway, thereby inhibiting APP cleavage by BACE1 and ultimately decreasing Aβ production. Concurrently, SDG also reduced APP and BACE1 levels, potentially through its regulation of MKP-1/ERK1/2.²⁹

Although 5×FAD mice do not develop neurofibrillary tangles,¹⁰ elevations in both phosphorylated tau levels and protein kinase activity have been reported and may be linked to Aβ deposition.^52–54 It has been well established that Aβ accelerates the hyperphosphorylation of tau protein, a process primarily mediated by the upregulation of CDK-5⁵⁵ and GSK-3β⁵⁶ activities. Notably, GSK-3β is involved in a complex interaction with Aβ. As well as being a key enzyme that drives tau phosphorylation, this kinase also increases the neurotoxic effects caused by hyperphosphorylated tau.^57,58 Further experimental evidence has revealed that MAPKs and GSK-3β are both implicated in the formation of paired helical filament-tau, a pathological hallmark, in the brains of individuals with AD.^59–61 In line with these findings, Aβ-elicited tau phosphorylation in neurons is dependent on the simultaneous activation of two distinct signaling molecules: MAPK and GSK-3β. Interestingly, SDG can antagonize Aβ-mediated MAPKs activation by increase MKP-1, thereby ultimately reducing phosphorylated tau.⁶²

Our results support that SDG improves cognitive function in 5×FAD mice by modulating APP metabolism, restoring memory-related protein levels, and reducing Aβ plaques. Concurrently, it inhibits tau phosphorylation and suppresses microglial/astroglial activation. These effects may be linked to SDG's enhancement of MKP-1 activity.

Footnotes

Acknowledgements

We are deeply grateful to Professor Yinghua Liu for her invaluable guidance and steadfast support throughout this research endeavor. Additionally, we acknowledge Guangzhou Medical University for offering the research platform that facilitated this work.

ORCID iD

Yinghua Liu

Ethical considerations

All animal experiments were performed in accordance with protocols approved by the Institutional Animal Care and Use Committee of Guangzhou Medical University.

Consent to participate

Not applicable

Author contribution(s)

Mei Chen: Data curation; Methodology; Visualization; Writing – original draft.

Qiuyu Xie: Methodology; Visualization; Writing – original draft.

Fangtai Yi: Methodology; Writing – original draft.

Jianfeng Ma: Methodology; Validation; Visualization.

Sitong Lu: Methodology.

Huijuan Ouyang: Methodology.

Yuan Qin: Supervision.

Shuo Yang: Writing – review & editing.

Wei Wei: Writing – review & editing.

Yinghua Liu: Funding acquisition; Project administration; Supervision; Writing – review & editing.

Funding

The authors disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: This work was supported by Natural Science Foundation of Guangdong Province (grant number 2019A1515010903 to YH Liu, for the routine reagents and materials of the research), High-level University Construction Fund of Guangdong Province (grant number YXY2021A05 to YH Liu, 06-410-2107217 to S Yang, for the routine reagents and materials of the research), Scientific and Technological Planning Program of Guangzhou (grant number 1201610286 to YH Liu, for the drugs, animals and their maintenance fees).

Declaration of conflicting interests

The authors declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Data availability statement

All data generated or analyzed during this study are included in this article. Further enquiries can be directed to the corresponding author.

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