β-Asarone Mediates the Alleviation of Neuroinflammation in Alzheimer’s Disease Via Modulation of the TREM2/PI3K/AKT Signaling Pathway

TL;DR

β-asarone reduces neuroinflammation in Alzheimer’s disease by regulating microglial activity through a specific signaling pathway.

Contribution

This study is the first to show that β-asarone alleviates AD pathology via the TREM2/PI3K/AKT/GSK3β signaling axis.

Findings

β-asarone improved cognitive function and reduced hippocampal damage in 3×Tg-AD mice.

The compound decreased Aβ deposition and Tau hyperphosphorylation in AD models.

β-asarone promotes microglial polarization from M1 to M2 phenotypes via TREM2/PI3K/AKT signaling.

Abstract

Neuroinflammation, driven by dysregulated microglial polarization, is a hallmark of Alzheimer’s disease (AD). Recently, the triggering receptor expressed on myeloid cells 2 (TREM2), a key regulator of microglial function, has emerged as a promising therapeutic target for AD. This study aimed to investigate the therapeutic potential and mechanism of action of the natural compound β-asarone in AD models. Our results demonstrate that β-asarone significantly improved cognitive function, reduced hippocampal neuronal damage, and decreased both Aβ deposition and Tau hyperphosphorylation in 3×Tg-AD mice. Mechanistically, β-asarone upregulated TREM2 expression, activated the PI3K/AKT pathway, and inhibited GSK3β activity, thereby promoting the polarization of microglia from the pro-inflammatory M1 phenotype toward the anti-inflammatory M2 phenotype and alleviating neuroinflammation. This study is the first to elucidate that β-asarone ameliorates AD pathology by modulating microglial polarization via the TREM2/PI3K/AKT/GSK3β signaling axis, providing experimental evidence supporting its potential as an immunomodulatory therapeutic agent for AD.

The online version contains supplementary material available at 10.1007/s10753-025-02435-w.

Figures

The positive effect of β-asarone on anti-neuroinflammation in 3×Tg-AD mice. A Representative immunofluorescence images of IBA1 in the hippocampal CA3 region (scale bars: 50 μm). B Representative immunofluorescence images of GFAP in the hippocampal CA3 region (scale bars: 50 μm). C Quantification of IBA1 immunofluorescence. D Quantification of GFAP immunofluorescence. E Hippocampal levels of proinflammatory cytokines (IL-1β, IL-8, and IL-6) were measured via ELISA. All data were presented as the mean ± SD (*n* = 3/group). Compared to the WT group, ^^*p* < 0.01; compared to the 3×Tg-AD group, ^#

Schematic diagram illustrating the mechanism by which β-asarone alleviates neuroinflammation by regulating microglial polarization via the TREM2/PI3K/AKT signaling pathway. β-asarone upregulates the microglial receptor TREM2, activating the PI3K/AKT signaling pathway. This activation inhibits GSK3β activity and NF-κB nuclear translocation, downregulates the expression of pro-inflammatory factors (TNF-α, IL-1β, IL-6), and promotes microglial polarization from the M1 to the M2 phenotype, ultimately alleviating neuroinflammation in AD

Full text

Introduction

Alzheimer’s disease (AD) represents a progressive neurodegenerative condition marked by the gradual impairment of cognition. Its hallmark pathological features include senile plaques caused by beta-amyloid (Amyloid β, Aβ) deposition and neurofibrillary tangles (NFTs) arising from the hyperphosphorylation of tau protein [1]. According to epidemiological research, the aging global population is driving a rising prevalence of AD. And it is expected that the number of patients will reach 152 million globally in 2050 [2], with the most significant increase in low-income and middle-income countries, which is a major burden to both society and families [3].

Neuroinflammation exacerbates the pathology of AD, including tau hyperphosphorylation and Aβ accumulation [4]. Under normal physiological conditions, microglia are responsible for immune surveillance and are crucial for maintaining cerebral homeostasis. During the early stage of AD, microglia mainly display an M2 phenotype that is considered neuroprotective. This phenotype facilitates the clearance of Aβ plaques and releases anti-inflammatory factors, including transforming growth factor (TGF)-β, interleukin (IL)−4, and IL-10 [5]. However, as the disease progresses, continuously activated microglia polarize toward the proinflammatory M1 phenotype, characterized by the upregulation of the expression of IBA1 and the substantial secretion of inflammatory factors such as IL-1β, IL-6, and tumor necrosis factor (TNF)-α, along with impaired phagocytic function, ultimately leading to the aggregation of Aβ and an intensification of neurodegenerative pathology [6, 7]. Therefore, elucidating the regulatory mechanisms governing the balance between microglial M1 and M2 polarization states during the progression of AD is of critical importance.

The triggering receptor expressed on myeloid cells 2 (TREM2), an immune receptor predominantly expressed in microglia, plays a critical and multifaceted role in the pathogenesis of AD [8]. Whole-genome association analyses have revealed that certain genetic variants (such as the R47H mutation) of the TREM2 gene greatly raise the risk for AD [9]. By regulating microglial phagocytosis, inflammatory responses, and cellular metabolism, TREM2 contributes to Aβ clearance and modulates neuroinflammation [10]. Recent evidence indicates that impaired TREM2 function compromises the ability of microglia to clear Aβ and exacerbates neuroinflammatory responses, whereas appropriate enhancement of its function may confer neuroprotective benefits [11, 12]. TREM2 can regulate microglial function by binding to ligands such as phospholipids, apolipoprotein E (APOE), lipopolysaccharide (LPS), and Aβ, activating downstream signaling pathways through DNAX-activation protein 12 (DAP12)/DAP10 [13, 14]. As the disease progresses, TREM2 expression increases, exhibiting stage-dependent effects that can exacerbate neuroinflammation in later stages while concomitantly promoting Aβ clearance and reducing inflammation, thereby providing neuroprotection in the early stages [15–17]. In AD models, TREM2 overexpression improves cognitive performance, suppresses microglial activation, and decreases neuronal loss [18, 19]. Therefore, targeting the TREM2 signaling pathway represents a key therapeutic approach to modulate microglial activity and enhance immune homeostasis in the context of neurodegenerative disorders.

β-asarone is the primary active constituent of the essential oil of the herb Acorus tatarinowii Schott, which is characterized by high volatility and lipophilicity, effectively crosses the blood-brain barrier, and has significant efficacy against AD. It provides neuroprotection by reducing Aβ-induced neurotoxicity, decreasing oxidative stress and mitochondrial dysfunction, and preventing neuronal death [20, 21]. Our earlier investigations showed that the essential oil extracted from this plant effectively improves cognitive dysfunction in both 3×Tg-AD mice and ICV-STZ rat models [22, 23]. Studies have indicated that β-asarone exerts neuroprotective effects through the regulation of PI3K/AKT, MAPK, and Nrf2/ARE signaling pathways [24]. However, the precise molecular mechanisms by which it exerts its therapeutic impact on AD have not yet been completely clarified.

In this study, 3×Tg-AD animals and wild-type (WT) control mice were utilized for the investigations and randomly assigned to various β-asarone treatment dosage groups, a donepezil-positive control group, and a model-control group for an eight-week pharmacological intervention. The Morris water maze (MWM) was employed to evaluate spatial learning and memory capabilities, alongside immunofluorescence, western blotting, and ELISA to carefully analyze alterations in Aβ deposition, tau protein phosphorylation, and the expression of neuroinflammation-related markers. To clarify the mechanism, we developed a model of LPS-induced inflammation in BV2 microglia, confirmed the pharmacodynamic mechanism of β-asarone via TREM2 gene overexpression and knockdown experiments, and systematically detailed the pathway of its action in modulating neuroinflammation through TREM2. This study offers fresh ideas for the treatment of AD.

Materials and Methods

Reagents and Antibodies

β-asarone (Lot #PRF2302144) was obtained from Chengdu Purefa Technology Development Co., Ltd. Donepezil (DNPZ) hydrochloride tablets (Lot #1705080) were obtained from Eisai Pharmaceutical Co., Ltd. LPS (Lot #S11060) was sourced from Shanghai Yuanye Biotechnology Co., Ltd. Antibody Aβ (Lot #sc-28365) was acquired from Santa Cruz Biotechnology. Antibodies Tau (Lot #46687), p-Tau (Lot #30505), protein kinase B (AKT; Lot #9292), and p-AKT (Lot #4060) were purchased from Cell Signaling Technology. Antibodies Glycogen synthase kinase-3β (GSK3β; Lot #A11731), phosphatidylinositol-3-kinase (PI3K; Lot #A4992), and p-PI3K (Lot #AP0427) were supplied by ABclonal. Antibodies IBA1 (Lot #ET1705-78), TREM2 (Lot #ER1918-04), CD86 (Lot #ET1606-50), CD206 (Lot #ET1702-04), p-GSK3β (Lot #ET1607-60), and GAPDH (Lot #ET1601-4) were purchased from HUABIO. Antibodies Glial fibrillary acidic protein (GFAP; Lot #YM3059), Goat anti-rabbit IgG (DyLight^®^ 800; Lot #RS23920), and Goat anti-mouse IgG (DyLight^®^ 800; Lot #RS23910) were purchased from ImmunoWay.

Drug Preparation

Aliquots of β-asarone with volumes of 75 µL, 150 µL, and 300 µL were mixed with an equivalent volume of the cosolvent Tween-80 and subsequently diluted with distilled water to a total volume of 50 mL, yielding final concentrations of 1.5, 3.0, and 6.0 mg/mL, respectively. DNPZ hydrochloride (10 mg) was dissolved in distilled water, and the volume was adjusted to 50 mL to obtain a concentration of 0.2 mg/mL. All solutions were then kept at 4 °C and shielded from the light.

Animal Grouping and Treatment

We used 3×Tg-AD mice (n = 70) and C57BL/6 mice (n = 14) in this study. All mice were nine months old, had a sex ratio of 1:1 (male: female), and weighed 25 ± 5 g. They were housed and bred in a nationally accredited animal research facility under standardized environmental conditions, which included a temperature maintained at 22 ± 1 °C, a 12-h light/dark cycle, 50–60% humidity, and 15–20 air changes per hour. The Animal Ethics and Welfare Committee approved the experimental protocol (Approval No. IACUC-20211227-04).

A total of 70 3×Tg-AD mice were randomly assigned into five experimental groups (n = 14/group): a model control group, three β-asarone treatment groups (15, 30, and 60 mg/kg), and a positive control group receiving donepezil (DNPZ, 2 mg/kg). An additional group of 14 WT mice served as the normal control. β-asarone doses were determined using the body surface area ratio conversion method and normalized for a 20 g mouse and a 70 kg person. The dosages used were as follows: the β-asarone groups received oral gavage at 15, 30, or 60 mg/kg, while the DNPZ group was given 2 mg/kg via the same route. Both the normal and model control groups received purified water. All treatments were administered once daily at a dosage of 0.1 mL per 10 g body weight over an eight-week period.

Morris Water Maze Test

The spatial learning and memory function of mice was examined via the MWM test (Harvard Apparatus, Smart-Mass00916s, Cornellà, Spain). The setup included a circular pool (120 mm in diameter, 50 mm in height) that was divided into four quadrants, each marked with a distinct visual cue near the entry point. Around 24 h before testing, all mice underwent a habituation swim session lasting 60 s. The hidden platform test was conducted over a period of five days, during which the mice were placed in the water facing the pool wall from random starting positions each day. The time taken to locate the platform (set in the first quadrant, 1 cm below the water surface) was recorded as escape latency (maximum 60 s). Any mouse that did not find the platform within 60 s was manually guided to the platform, and the latency was recorded as 60 s. On the sixth day, a spatial probing test was conducted with the platform removed. The swimming path, the number of crossings over the previous platform location, and the percentage of time spent in the target quadrant within 60 s were recorded.

Brain Tissue Collection

Upon anesthesia and blood collection, the mice were promptly sacrificed for rapid extraction of brain tissues, followed by careful dissection of the hippocampal regions. The whole brain after perfusion was fixed with 4% paraformaldehyde and then prepared into sections for hematoxylin-eosin (HE) staining, Nissl staining, and immunofluorescence staining. A quarter of the hippocampus of fresh brain tissue was taken for transcriptome sequencing, and the remaining tissue was preserved at − 80 °C for future molecular analysis.

Preparation of Brain Tissue Slices and Pathological Staining

The mouse brain tissues were embedded in paraffin and cut into 4 μm slices after being fixed for 24 h at 4 °C in 4% paraformaldehyde. The HE staining process included xylene dewaxing, graded ethanol hydration, hematoxylin staining (5 min), differentiation in 0.5% hydrochloric acid-alcohol, bluing in 0.5% ammonia water, eosin counterstaining (2 min), graded dehydration, and xylene clearing. The eosin counterstaining step in Nissl staining was replaced with staining in toluidine blue for 30 min. After staining, the sections were coverslipped using neutral balsam and observed under a light microscope for histological assessment of the hippocampal CA3 region.

Transcriptomics

Hippocampal tissues from three randomly selected mice in each of the control, model, and medium-dose β-asarone groups were subjected to RNA extraction and transcriptome sequencing by LC-Bio Technology Co., Ltd. (Hangzhou, China). Differentially expressed genes (DEGs) were identified based on the criteria of |Fold Change| ≥ 1.5 and p < 0.05. Gene Ontology (GO) functional annotation and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis were conducted using the LC-Bio Cloud Platform (www.omicstudio.cn), and volcano plots of the DEGs were generated using the Bioinformatics Platform (www.bioinformatics.com.cn).

Cell Culture

The BV2 microglial cell line (Cyagen Biosciences, Shanghai, China) was cultured in high-glucose Dulbecco’s modified Eagle’s medium containing 10% fetal bovine serum and 1% penicillin-streptomycin. Cells were incubated at 37 °C in a humidified atmosphere of 5% CO_2_ with 95% humidity. Subculturing was carried out upon reaching 80–90% confluency.

Lentiviral Transfection

Lentiviral vectors (OE-TREM2, OE-NC, sh-TREM2, and sh-NC) were constructed and packaged by GeneChem (Shanghai, China). BV2 cells in the logarithmic growth phase were seeded in six-well plates at a density of 5 × 10^5^ cells per well and transfected according to the manufacturer’s (GeneChem) protocol. Transfection efficiency (> 80%) was evaluated by fluorescence microscopy 72 h after transfection, and the optimal multiplicity of infection (MOI = 20) was determined. Stable transfectants were selected using 2 µg/mL puromycin for five days. Then cell viability was evaluated, and TREM2 protein and mRNA expression levels were analyzed.

CCK-8 Assay

BV2 cells in the logarithmic growth phase were dissociated and adjusted to a density of 1 × 10^5^ cells/mL and then seeded in 96-well plates (100 µL/well). When the cells reached 50–70% confluence, they were treated with LPS (0, 0.2, 0.4, 0.6, 0.8, 1, 1.2, 1.4, or 1.6 µg/mL) or β-asarone (0, 1, 2, 4, 8, 16, 32, 64, or 128 µM) for 24 h, with six replicate wells for each group. After treatment, each well received 10 µL of CCK-8 reagent and was incubated for 1 h at 37 °C, after which absorbance readings were taken at 450 nm.

Western Blotting

Protein extraction from hippocampal tissues and BV2 cells was performed using RIPA buffer containing protease and phosphatase inhibitors. The supernatant was collected after centrifugation at 12,000 rpm for 15 min at 4 °C, and the BCA assay was used to determine the protein concentration. Equal amounts of protein were separated by SDS-PAGE and electrophoretically transferred to PVDF membranes. After blocking the membranes with 5% BSA for 1 h at room temperature, they were incubated with primary antibodies at 4 °C for the whole night. After TBST washes, membranes were incubated with HRP-conjugated secondary antibodies for 1 h at ambient temperature. An Odyssey CLx imaging system was used to visualize the protein bands, and ImageJ software was used for densitometric analysis and quantification.

Immunofluorescence

Paraffin sections were rehydrated using a graded series of ethanol after being deparaffinized with xylene. Microwave heating in 0.01 M sodium citrate buffer (pH 6.0) for 7 min was used to retrieve the antigen. Sections were blocked with 3% BSA for an hour at room temperature after phosphate-buffered saline (PBS) washing, and they were then incubated with primary antibodies for the whole night at 4 °C. Following several PBS washes the next day, the slices were exposed to Alexa Fluor 647-conjugated secondary antibodies and DAPI for 1 h at 37 °C under light-protected conditions. After a final series of washes in PBS, the tissue sections were coverslipped using an anti-fade mounting medium. Imaging was performed using an inverted fluorescence microscope to capture the fluorescent signals (Leica DMI3000B, Germany).

The BV2 cells were plated onto coverslips positioned in 24-well culture plates at an initial density of 1 × 10^5^ cells per well and cultured overnight to ensure adhesion. After being treated with LPS or β-asarone, the cells were fixed with 4% paraformaldehyde for 15 min and permeabilized with 0.1% Triton X-100 for 10 min. Then they were treated with primary antibodies overnight at 4 °C after 1 h of 3% BSA blocking. Subsequently, they were incubated with Alexa Fluor 488-conjugated or 647-conjugated secondary antibodies and DAPI for 1 h at room temperature under light-protected conditions. Finally, the coverslips were mounted using anti-fade medium and visualized with a laser scanning confocal microscope (ZEISS LSM880, Germany).

ELISA

The levels of inflammatory factors in mouse hippocampal tissue and BV2 cell culture supernatants were measured strictly following the instructions provided with the ELISA kit. The optical density of the reaction mixture was recorded at 450 nm with a microplate reader (Thermo Fisher Scientific, USA), and all data were analyzed to calculate the concentration based on standard curves. Commercial ELISA kits for mouse IL-1β, IL-8, IL-6, and TNF-α were sourced from Quanzhou Ruixin Biotechnology Co., Ltd. (Quanzhou, China).

Statistical Analysis

Statistical analyses were conducted using GraphPad Prism software (version 8.0.2). Data are expressed as mean ± standard deviation (SD). Comparisons across multiple groups were performed using one-way ANOVA followed by Tukey’s test for post hoc analysis. For comparisons involving two groups, unpaired Student’s t-tests were applied when normality and homogeneity of variance assumptions were satisfied; otherwise, Welch’s corrected t-tests were applied. The statistical significance of all results was considered to be p < 0.05.

Results

β-Asarone Ameliorates Cognitive Deficits in 3×Tg-AD Mice

During the five-day navigation training phase of the MWM test, the 3×Tg-AD mice displayed noticeably prolonged escape latencies relative to WT controls, with no notable enhancement during training. In contrast, the β-asarone and DNPZ treatment groups exhibited markedly reduced escape latencies versus the untreated 3×Tg-AD group (Fig. 1A). During the probe trial session, the 3×Tg-AD group demonstrated impaired spatial memory, as indicated by reduced crossings over the platform, diminished time spent in the target quadrant, and longer path lengths traveled. Both the β-asarone and DNPZ treatments considerably improved these deficiencies, as indicated by increased platform crossings, longer target quadrant durations, and shorter swimming paths (Fig. 1B-D).Fig. 1β-asarone improves cognitive function and pathological damage in 3×Tg-AD mice. A Escape latency in the Morris water maze test. B Number of platform crossings (n = 14/group). C Percentage of time spent in the target quadrant (n = 14/group). D Representative swimming trajectories. E HE staining of the hippocampal CA3 region (scale bars: 20 μm). F Nissl staining of the hippocampal CA3 region (scale bars: 20 μm). G Representative immunofluorescence images of Aβ_1−42_ in the hippocampal CA3 region (scale bars: 50 μm). H Quantification of Aβ_1−42_ immunofluorescence. I Expression levels of the Tau and p-Tau proteins in hippocampal tissues were analyzed by western blotting (n = 3/group). All data were presented as the mean ± SD. Compared to the WT group, ^**^p < 0.01 and ^*^p < 0.001; compared to the 3×Tg-AD group, ^#^p < 0.05 and ^##^p < 0.01

β-Asarone Improves Pathological Changes in 3×Tg-AD Mice

Histopathological evaluation of hippocampal CA3 neuronal integrity in 3×Tg-AD mice was examined via HE and Nissl staining. HE staining revealed that neurons in the hippocampal CA3 region of WT mice maintained a normal morphology, were densely arranged with round nuclei and distinct nucleoli, and showed no significant pathological alterations. In contrast, hippocampal tissue from 3×Tg-AD mice showed disrupted neuronal architecture, decreased cellular density, and hallmark degenerative changes such as cell body shrinkage, nuclear pyknosis, and intensely stained cytoplasm. After treatment with various doses of β-asarone and DNPZ, these pathological changes were significantly ameliorated, evidenced by regular neuronal arrangement and a decrease in cell shrinkage and nuclear condensation (Fig. 1E). Nissl staining confirmed that pyramidal cells within the hippocampal CA3 of WT mice maintained an intact morphology, with abundant and evenly distributed Nissl bodies, prominently stained nuclei, and well-preserved structures. The 3×Tg-AD group presented marked Nissl body dissolution, neuronal loss, and deeply stained nuclei, indicating neuronal dysfunction. In the β-asarone-treated and DNPZ-treated groups, the number of Nissl bodies was significantly restored, the cellular morphology closely resembled that of the normal controls, and the nucleolar structure improved considerably (Fig. 1F).

Immunofluorescence staining revealed significantly increased Aβ_1−42_ expression within the hippocampal CA3 area of 3×Tg-AD mice. Administration of β-asarone and DNPZ resulted in a pronounced reduction in Aβ_1−42_ deposition (Fig. 1G-H). Abnormal tau protein phosphorylation leads to the formation of NFTs, one of the pathological processes occurring in early AD stages. In contrast to WT controls, 3×Tg-AD mice showed a markedly raised p-Tau/Tau ratio, indicating elevated phosphorylation levels, whereas the administration of β-asarone and DNPZ effectively attenuated this abnormality (Fig. 1I).

The Anti-Neuroinflammatory Effect of β-Asarone in 3×Tg-AD Mice

The abnormal activation of astrocytes and microglia is a significant contributor to the pathological development of AD. They exacerbate neurodegeneration by releasing proinflammatory factors and neurotoxic substances. Relative to WT mice, the 3×Tg-AD mice displayed markedly stronger immunofluorescence signals of IBA1 and the astrocytic marker GFAP in the CA3 region of the hippocampus (Fig. 2A-D). Following oral administration of β-asarone or DNPZ, the expression levels of IBA1 and GFAP decreased considerably, suggesting that β-asarone may alleviate neuroinflammation by influencing the activation states of glial cells.Fig. 2. The positive effect of β-asarone on anti-neuroinflammation in 3×Tg-AD mice. A Representative immunofluorescence images of IBA1 in the hippocampal CA3 region (scale bars: 50 μm). B Representative immunofluorescence images of GFAP in the hippocampal CA3 region (scale bars: 50 μm). C Quantification of IBA1 immunofluorescence. D Quantification of GFAP immunofluorescence. E Hippocampal levels of proinflammatory cytokines (IL-1β, IL-8, and IL-6) were measured via ELISA. All data were presented as the mean ± SD (n = 3/group). Compared to the WT group, ^****^p < 0.01; compared to the 3×Tg-AD group, ^##^p < 0.01

To further examine neuroinflammatory reactions, we measured hippocampal concentrations of IL-1β, IL-8, and IL-6 via ELISA. Relative to the WT controls, the 3×Tg-AD mice showed considerably increased expression of these cytokines. Treatment with β-asarone or DNPZ considerably decreased their expression in 3×Tg-AD mice (Fig. 2E), demonstrating the anti-neuroinflammatory effects of β-asarone.

β-Asarone Promotes M1-to-M2 Polarization in BV2 Cells during LPS-Induced Inflammatory Responses

Microglial activation serves as a critical pathological manifestation of neuroinflammation. We established a well-characterized inflammatory model in BV2 cells using 1 µg/mL LPS [25, 26]. Our results indicate that β-asarone (8, 16, and 32 µM) inhibited LPS-induced microglial activation in a concentration-dependent manner (Figs. 3A-D and 4A). Analysis of inflammatory mediators in the cell culture supernatants revealed that exposure to LPS led to a pronounced elevation in the release of proinflammatory cytokines (TNF-α, IL-1β, and IL-6), whereas β-asarone treatment substantially decreased these levels (Fig. 3E). Compared to the control conditions, LPS stimulation significantly upregulated the M1 marker CD86 while downregulating the M2 marker CD206 in BV2 cells; these effects were reversed by β-asarone treatment (Fig. 3F-H). These findings were confirmed by qRT-PCR and flow cytometry analyses (Fig. S1).Fig. 3β-asarone inhibits the LPS-induced inflammatory response in BV2 cells. A CCK8 assay was performed to detect the effect of LPS on the viability of BV2 cells (n = 6/group). B CCK8 was used to detect the effect of β-asarone on the viability of normal BV2 cells (n = 6/group). C The CCK-8 assay determined the effect of β-asarone on the viability of the BV2 cell inflammation model (n = 6/group). D Effect of β-asarone on the morphology of BV2 cells (scale bars: 50 μm). E The levels of inflammatory cytokines (TNF-α, IL-1β, and IL-6) in the BV2 cell culture supernatant were measured by ELISA. F Representative immunofluorescence images of CD86 and CD206 in BV2 cells (scale bars: 10 μm). G Quantification of CD86 immunofluorescence. H Quantification of CD206 immunofluorescence. All data are presented as the mean ± SD (n = 3/group). Compared to the control group, ^****^p < 0.01, ^***^p < 0.001, and ns indicates no statistical significance; compared to the LPS group, ^#^p < 0.05, ^##^p < 0.01, and ^###^p < 0.001Fig. 4β-asarone reverses the LPS-induced downregulation of TREM2 in BV2 cells. A Western blotting was performed to assess the expression of TREM2 and IBA1 in BV2 cells. B Representative immunofluorescence images of TREM2 in BV2 cells (scale bars: 10 μm). C Quantification of TREM2 immunofluorescence. All data are presented as the mean ± SD (n = 3/group). Compared to the control group, ^^p < 0.01; compared to the LPS group, ^#^p < 0.05, ^##^p < 0.01, and ^###^p < 0.001

β-Asarone Reverses the LPS-Induced Downregulation of TREM2 in BV2 Cells

TREM2 is specifically expressed on microglia and can serve as a new target for the treatment of AD by regulating microglial phagocytosis, the inflammatory response, and metabolic reprogramming. In this study, we observed that LPS stimulation decreased TREM2 expression in BV2 cells, while treatment with β-asarone significantly upregulated its expression (Fig. 4A). And the immunofluorescence staining results were consistent with this finding (Fig. 4B-C).

β-Asarone Attenuates LPS-Induced Inflammatory Responses in BV2 Cells through TREM2-Mediated Mechanisms

To elucidate the involvement of TREM2 in modulating neuroinflammatory processes in AD, we performed lentivirus-mediated overexpression and knockdown of TREM2 in BV2 microglial cells (Figs. S2 and S3). Western blotting and immunofluorescence results confirmed that LPS significantly decreased TREM2 expression and that β-asarone significantly restored it. In TREM2-knockdown cells, TREM2 expression was clearly elevated in the LPS + β-asarone group relative to the LPS-only group, but β-asarone did not further considerably increase TREM2 expression in TREM2-overexpressing cells (Fig. 5A-B and E).Fig. 5β-asarone attenuates LPS-induced inflammatory responses in BV2 cells through TREM2-mediated mechanisms. A Western blotting was performed to detect the protein expression of IBA1 and TRME2 in BV2 cells after they were infected with lentivirus. B Representative images of TREM2 immunofluorescence in BV2 cells after lentivirus treatment (scale bars: 10 μm). C ELISA was performed to measure the levels of the inflammatory factors TNF-α, IL-1β, and IL-6 in the culture supernatant of BV2 cells after lentivirus infection. D Representative images of CD86 and CD206 immunofluorescence in BV2 cells after lentivirus treatment (scale bars: 10 μm). E Quantification of TREM2 immunofluorescence. F Quantification of CD86 immunofluorescence. G Quantification of CD206 immunofluorescence. All data are presented as the mean ± SD (n = 3/group). Compared to the NC group, ^aa^p < 0.01 and ^aaa^p < 0.001; compared to the LPS group, ^b^p < 0.05, ^bb^p < 0.01, and ^bbb^p < 0.001; compared to the LPS + β-asarone group, ^c^p < 0.05, ^cc^p < 0.01, and ^ccc^p < 0.001; compared to the sh-TREM2 + LPS group, ^d^p < 0.05 and ^dd^p < 0.01. NC: negative control; β-asarone: 32 µM β-asarone; sh-TREM2: TREM2 knockdown; OE-TREM2: TREM2 overexpression

The ELISA results revealed that TREM2 knockdown elevated the secretion of TNF-α, IL-1β, and IL-6 in supernatants and partially attenuated the protective effects of β-asarone, whereas the overexpression of TREM2 moderately reduced inflammation levels and enhanced the effects of β-asarone (Fig. 5C). Immunofluorescence staining verified that knockdown of TREM2 in BV2 cells led to upregulated expression of CD86 and downregulated expression of CD206, while TREM2 overexpression reversed these effects (Fig. 5D and F-G). These results indicate that β-asarone reduces the secretion of pro-inflammatory factors by upregulating the expression of TREM2, inhibits microglial M1 polarization, and alleviates inflammatory responses.

These findings demonstrate that β-asarone effectively increases TREM2 expression, reducing proinflammatory factor release, inhibiting microglial M1 polarization, and alleviating inflammatory responses. Additionally, β-asarone partially reverses the pathological changes induced by TREM2 knockdown, although its therapeutic efficacy is attenuated in TREM2-knockdown models, whereas TREM2 overexpression moderately increases the effects of β-asarone.

β-Asarone Confers Anti-Neuroinflammatory Action through the TREM2/PI3K/AKT Pathway

Transcriptome sequencing was carried out on hippocampal tissue isolated from WT mice, 3×Tg-AD mice, and 3×Tg-AD mice treated with 30 mg/kg β-asarone. DEGs were defined as those showing |fold change (FC)| ≥ 1.5 and p < 0.05. Compared to the WT group, the 3×Tg-AD group had 344 DEGs (119 downregulated and 225 upregulated). After β-asarone treatment, the expression of 708 genes (525 downregulated and 183 upregulated) was altered (Fig. 6A), among which 178 were shared DEGs (Fig. 6B). We conducted GO and KEGG enrichment analyses on the 178 DEGs obtained from the RNA sequencing of hippocampal tissues in the aforementioned mice. These genes are predominantly engaged in key biological processes such as cellular adhesion, signal transmission, and lipid metabolic pathways. They are also localized to essential cellular structures, including the cytoplasm, plasma membrane, and endoplasmic reticulum. Molecular function analysis indicated involvement in protein binding, metal ion binding, and ATP binding. These genes exhibited significant enrichment in key signaling pathways, including the PI3K-Akt, cGMP-PKG, and TGF-β pathways (Fig. 6C). Western blotting analysis revealed that LPS treatment significantly inhibited PI3K, AKT, and GSK3β phosphorylation in BV2 cells, whereas β-asarone reversed this inhibition (Fig. 6D). Further experiments revealed that TREM2 knockdown reduced the phosphorylation levels within the PI3K/AKT pathway and reduced the effects of β-asarone, whereas TREM2 overexpression activated this signaling pathway (Fig. 6E).Fig. 6β-asarone confers anti-neuroinflammatory action through the TREM2/PI3K/AKT pathway. A DEGs in 3×Tg-AD mice were identified by transcriptomic sequencing. B The Venn diagram illustrates overlapping DEGs before and after β-asarone treatment. C GO and KEGG functional enrichment analyses of the DEGs identified via transcriptomic sequencing. D Western blotting analysis of the expression of PI3K/AKT pathway-related proteins in BV2 cells. E Western blotting analysis was performed to detect the expression of PI3K/AKT pathway-related proteins in BV2 cells after lentiviral treatment. All data are presented as the mean ± SD (n = 3/group). Compared to the control group, ^****^p < 0.01 and ^*****^p < 0.001; compared to the LPS group, ^#^p < 0.05, ^##^p < 0.01, and ^###^p < 0.001. Compared to the NC group, ^a^p < 0.05, ^aa^p < 0.01, and ^aaa^p < 0.001; compared to the LPS group, ^b^p < 0.05, ^bb^p < 0.01, and ^bbb^p < 0.001; compared to the LPS + β-asarone group, ^cc^p < 0.01 and ^ccc^p < 0.001; compared to the sh-TREM2 + LPS group, ^d^p < 0.05. NC: negative control; β-asarone: 32 µM β-asarone; sh-TREM2: TREM2 knockdown; OE-TREM2: TREM2 overexpression

Discussion

Alzheimer’s disease remains a formidable challenge in neurodegenerative disorders, with neuroinflammation orchestrated by microglia playing a pivotal role in its pathogenesis [27]. This study systematically evaluated the therapeutic potential and underlying mechanisms of β-asarone, a natural bioactive compound, in both the 3×Tg-AD triple-transgenic mouse model of AD and the LPS-induced BV2 microglial inflammation model. Our results collectively demonstrate that β-asarone significantly ameliorates cognitive deficits, alleviates neuropathological injury, and exerts robust anti-neuroinflammatory effects in AD model mice. Importantly, we reveal for the first time that these beneficial outcomes are mediated, at least in part, by the upregulation of TREM2, which modulates the PI3K/AKT signaling pathway and promotes the polarization of microglia from the pro-inflammatory M1 phenotype toward the anti-inflammatory M2 phenotype.

Using the MWM test, we demonstrated that β-asarone treatment significantly shortened escape latency and improved spatial memory retrieval in 3×Tg-AD mice, as evidenced by increased platform crossings, prolonged time spent in the target quadrant, and more efficient swimming paths. These behavioral outcomes are consistent with previous reports indicating that β-asarone ameliorates learning and memory deficits in transgenic AD models. Notably, the efficacy of β-asarone was comparable to that of DNPZ, a clinically used AD drug, further supporting its potential as a therapeutic candidate for AD.

Improvements in cognitive function are often accompanied by the restoration of neural pathology. Histological analyses revealed that β-asarone effectively alleviated degenerative changes in the hippocampal CA3 region of 3×Tg-AD mice, including disorganized neuronal alignment, reduced cell density, and Nissl body dissolution. Nissl bodies, aggregates of rough endoplasmic reticulum and free ribosomes in neurons, serve as morphological indicators of protein synthesis capacity and metabolic activity [28, 29]. The preservation of Nissl bodies by β-asarone suggests its role in maintaining neuronal structural integrity and functional competence. At the molecular level, β-asarone significantly reduced Aβ_1−42_ deposition and suppressed Tau hyperphosphorylation in the hippocampus. These findings suggest that β-asarone concurrently modulates key pathological cascades associated with both the “Aβ hypothesis” and “Tau hypothesis” of AD. The reduction in Aβ deposition may be attributed to the regulation of amyloid precursor protein processing and enhanced Aβ clearance, which has been partially validated in prior studies [30, 31]. Meanwhile, the inhibition of Tau phosphorylation may involve the regulation of kinase (e.g., GSK3β) and phosphatase activities [32, 33], and the observed modulation of the PI3K/AKT/GSK3β pathway in this study offers a plausible mechanistic explanation.

Neuroinflammation is recognized as a key factor in the pathogenesis of AD, acting alongside and even synergistically with Aβ and Tau pathologies. In the AD brain, aberrant activation of microglia and astrocytes leads to the release of numerous pro-inflammatory cytokines, such as IL-1β, IL-6, and TNF-α [34]. These cytokines not only directly damage neurons but also exacerbate Aβ deposition and Tau phosphorylation, thereby establishing a vicious cycle [35, 36]. Our results clearly show that β-asarone has strong anti-neuroinflammatory effects. In vivo, β-asarone significantly reduced the overexpression of the astrocytic marker GFAP and the microglial marker IBA1 in the hippocampus of 3×Tg-AD mice, while also suppressing elevated levels of pro-inflammatory cytokines IL-1β, IL-6, and IL-8. This result aligns with in vitro that β-asarone inhibits the production of pro-inflammatory mediators in LPS-activated BV2 microglial cells.

To further investigate its anti-neuroinflammatory mechanisms, we employed an LPS-induced BV2 microglial inflammation model. Microglia can polarize into distinct functional phenotypes in response to varying microenvironmental stimuli. The classically activated M1 phenotype (expressing CD86) predominantly secretes pro-inflammatory factors and exhibits neurotoxic effects, while the alternatively activated M2 phenotype (expressing CD206) releases anti-inflammatory and neurotrophic factors that facilitate tissue repair and the resolution of inflammation [37–39]. Our findings indicate that β-asarone suppressed LPS-induced BV2 cell activation in a concentration-dependent manner and significantly reduced the levels of TNF-α, IL-1β, and IL-6 in the culture supernatant. More importantly, β-asarone treatment downregulated the expression of the M1 marker CD86 while upregulating the M2 marker CD206. This finding is crucial, as it suggests that the anti-inflammatory effect of β-asarone is not merely a suppression of microglial activity but rather an active reprogramming that shifts microglia from the neurotoxic M1 phenotype toward the neuroprotective M2 phenotype, thereby fundamentally modulating the destructive course of neuroinflammation.

To elucidate the mechanism by which β-asarone reprograms microglial phenotypes, we focused on TREM2. TREM2 is an immune receptor predominantly expressed in microglia, and loss-of-function mutations in TREM2 have been identified through genome-wide association studies as significant genetic risk factors for AD [40, 41]. By regulating microglial phagocytosis, inflammatory responses, and cellular metabolism, TREM2 plays a multifaceted protective role in AD pathogenesis [42]. Our experiments showed that LPS stimulation significantly suppressed TREM2 expression in BV2 cells, whereas β-asarone treatment effectively reversed this trend and markedly upregulated TREM2 protein levels. To verify the central role of TREM2 in β-asarone’s mechanism of action, we employed lentivirus-mediated knockdown and overexpression techniques. Gain- and loss-of-function assays provided compelling evidence: when TREM2 was knocked down, the inhibitory effect of β-asarone on pro-inflammatory cytokine release was partially attenuated, and its promotion of M1-to-M2 phenotype transition was also weakened. Conversely, in TREM2-overexpressing cells, the anti-inflammatory effect of β-asarone was further enhanced.

Finally, to elucidate the downstream signaling events of TREM2, we performed transcriptomic sequencing and bioinformatic analysis on mouse hippocampal tissues. We discovered that the differentially expressed genes subsequent to β-asarone treatment were markedly enriched in the PI3K/AKT signaling pathway. The PI3K/AKT pathway is a crucial regulator of cell survival and metabolism, and emerging evidence has highlighted its central role in modulating inflammatory responses and microglial polarization [43–47]. Activation of AKT leads to phosphorylation and inhibition of its downstream target GSK3β, whose activity is closely linked to the production of pro-inflammatory factors and M1 polarization [48–50]. Our in vitro experiments validated this bioinformatic prediction. Western blot analysis demonstrated that LPS treatment markedly inhibited the phosphorylation of PI3K, AKT, and GSK3β in BV2 cells, signifying a suppression of the pathway’s activity. In contrast, β-asarone treatment effectively restored the phosphorylation status of the PI3K/AKT/GSK3β signaling axis. Importantly, this effect was also dependent on TREM2; knocking down TREM2 suppressed PI3K/AKT pathway activity and attenuated the activating effect of β-asarone, whereas TREM2 overexpression enhanced pathway activation. This mechanism aligns with previous findings that β-asarone inhibits autophagy in a Parkinson’s disease-related depression model by activating the PI3K/AKT/mTOR pathway [51], suggesting that PI3K/AKT signaling may represent a common central node through which β-asarone exerts its neuroprotective effects.

This study confirmed that β-asarone upregulates TREM2 expression in microglia and activates its downstream PI3K/AKT signaling pathway. Notably, recent research has identified Cyclophilin A (CypA) as a novel endogenous high-affinity ligand for TREM2. Ji et al. [52] demonstrated through binding assays that CypA specifically interacts with the Pro^144^ residue in the extracellular domain of TREM2, directly activating downstream signals such as Syk and PI3K/AKT, thereby modulating the production of inflammatory cytokines. This discovery establishes the CypA-TREM2 axis as a crucial component in the immunoregulation of myeloid cells. We further integrate the latest research by Pashaei et al. [53], which details the multifaceted role of CypA in AD. Beyond its established pro-inflammatory effects, it exacerbates neuroinflammation, disrupts the blood-brain barrier, and promotes Aβ deposition and Tau hyperphosphorylation, collectively driving neurodegenerative pathology, and this study crucially reveals that CypA exhibits a “bidirectional regulatory” capacity over TREM2 expression. Exogenous CypA can upregulate TREM2, while abnormal intracellular accumulation may suppress its expression via negative feedback mechanisms. This dynamic balance is likely closely associated with different disease stages and inflammatory microenvironment states. Integrating our findings, we speculate that the anti-neuroinflammatory effects of β-asarone may be partially achieved by influencing this precise CypA-TREM2 regulatory axis. Future research warrants in-depth exploration of whether β-asarone directly intervenes in CypA expression or its interaction with TREM2, which would provide a novel perspective for elucidating its multi-target neuroprotective mechanisms.

Although this study provides compelling evidence that β-asarone exerts its anti-neuroinflammatory effects via upregulation of the TREM2/PI3K/AKT signaling axis, several limitations warrant consideration. First, the precise mechanism by which β-asarone regulates TREM2 remains to be fully elucidated. It is currently unclear whether it acts by directly modulating the TREM2 promoter activity, influencing upstream transcriptional regulators, or affecting post-transcriptional mechanisms. Furthermore, it will be important to investigate whether, beyond the PI3K/AKT pathway, other signaling cascades downstream of TREM2 (such as those involving SYK or AMPK) also contribute to the observed shifts in microglial polarization. Finally, the experimental models employed present inherent constraints. The LPS-induced BV2 cell model, while valuable, may not fully recapitulate the chronic, multi-faceted neuroinflammatory milieu of AD. Taking these points into consideration, our future research will aim to establish a crucial foundation for developing TREM2-targeted therapeutic strategies against AD by providing more precise mechanistic data.

Conclusion

In summary, this study systematically elucidates the therapeutic effects and underlying mechanisms of β-asarone in AD models across multiple levels, ranging from whole organisms to molecular pathways. We have not only confirmed its significant efficacy in improving cognitive function and mitigating neuropathology but also innovatively uncovered a novel mechanism centered on TREM2 and mediated through the PI3K/AKT signaling pathway (Fig. 7). This discovery connects the therapeutic potential of a natural compound with key immune regulatory mechanisms in AD, therefore offering a novel conceptual framework for comprehending the neuroprotective effects of β-asarone.

Fig. 7. Schematic diagram illustrating the mechanism by which β-asarone alleviates neuroinflammation by regulating microglial polarization via the TREM2/PI3K/AKT signaling pathway. β-asarone upregulates the microglial receptor TREM2, activating the PI3K/AKT signaling pathway. This activation inhibits GSK3β activity and NF-κB nuclear translocation, downregulates the expression of pro-inflammatory factors (TNF-α, IL-1β, IL-6), and promotes microglial polarization from the M1 to the M2 phenotype, ultimately alleviating neuroinflammation in AD

Supplementary Information

Below is the link to the electronic supplementary material.

Supplementary File 1 (DOCX 1.58 MB)

Supplementary File 2 (PDF 3.42 MB)