Olfactory Mucosa Mesenchymal Stem Cell–Derived Exosomes Enhance Microglia M2 Polarization via the FGFR1/PLCγ1 Axis to Alleviate Alzheimer’s Disease

Abstract

This study aims to investigate the effect of exosomes derived from olfactory mucosa mesenchymal stem cells (OM-MSCs-Exo) on microglial polarization and its potential therapeutic role in Alzheimer’s disease (AD). OM-MSCs-Exo were isolated and purified from the mice olfactory mucosa, followed by phenotypic characterization. Proteins transferred by OM-MSCs-Exo were screened using proteomic analysis. The AD model was established in microglial cells and mice with Aβ_1–42. Immunofluorescence and biochemical assays were employed to assess the impact of OM-MSCs-Exo and its secreted protein FGFR1 on microglial polarization. Protein–protein interactions and immunoprecipitation were used to identify the target proteins of FGFR1 in microglial cells. Additionally, the effects of OM-MSCs-Exo-induced microglial polarization on neuronal inflammation and cognitive function in mice were evaluated. OM-MSCs-Exo were successfully isolated and purified. FGFR1 was significantly upregulated in OM-MSCs-Exo compared to OM-MSCs. Aβ_1–42 induced M1 polarization and suppressed M2 polarization of microglia, which was reversed by OM-MSCs-Exo. FGFR1 overexpression in OM-MSCs-Exo further enhanced M2 polarization in microglial cells. Phospholipase C gamma 1 (PLCγ1) was identified as the target of FGFR1, and knocking down PLCγ1 reversed the effects of FGFR1-overexpressing OM-MSCs-Exo. OM-MSCs-Exo alleviated cognitive decline and neuroinflammation in AD mice, with FGFR1 overexpression further enhancing these effects. OM-MSCs-Exo promote M2 polarization of microglia in AD mice through the FGFR1/PLCγ1 pathway, alleviating neuronal inflammation and cognitive dysfunction.

Supplementary Information

The online version contains supplementary material available at 10.1007/s12035-026-05797-w.

Introduction

Alzheimer’s disease (AD) is a chronic neurodegenerative disorder characterized by progressive cognitive and functional decline [1]. It is the most common form of dementia, accounting for approximately two-thirds of all cases worldwide [2]. The core pathological features of AD include the deposition of beta-amyloid (Aβ) plaques and hyperphosphorylation of tau tangles [3]. Currently, available treatments can only alleviate symptoms, with no cure [4]. Developing novel therapeutic strategies for AD remains an urgent challenge.

Mesenchymal stem cells (MSCs), particularly those derived from olfactory mucosa, have gained attention as potential candidates for AD modeling and therapy due to their regenerative potential and ease of accessibility [5]. Transplantation of olfactory mucosa–derived MSCs (OM-MSCs) has shown promising results in improving cognitive function and motor behavior in AD mice. Olfactory dysfunction, an early clinical symptom of AD, suggests a potential link between olfactory pathway integrity and disease progression [6]. OM-MSCs, which can be relatively accessible from the nasal mucosa, possess neural differentiation potential and represent a clinically relevant cell source for investigating AD-related repair mechanisms [7–10]. Furthermore, OM-MSCs promote the polarization of BV2 microglial cells from the pro-inflammatory M1 phenotype to the anti-inflammatory M2 phenotype and facilitate the clearance of Aβ in AD neuron cell models [11]. However, the underlying mechanisms by which OM-MSCs exert these effects in AD remain poorly understood.

In recent years, microglia transplantation and exosome (Exo) infusion have become popular areas of research in AD treatment [12]. The immunomodulatory properties of MSCs are largely attributed to their release of extracellular vesicles (EVs), including Exo and microvesicles [13]. Exo are nanoscale membrane vesicles that transfer bioactive molecules between cells and regulate various biological processes under both physiological and pathological conditions [14]. Exo have emerged as a novel platform for treating a range of diseases, including cancer, neurological disorders, cardiovascular diseases, and immune-related conditions [15]. MSCs have been used to treat neuronal injuries and neurodegenerative diseases, with their therapeutic potential attributed to the secretion of paracrine factors that promote tissue repair [16, 17]. Exo, as key components of paracrine signaling, play a significant role in these effects [18]. Intranasal administration of MSC-derived EVs has shown potential in inducing immunomodulation and neuroprotection in AD [19]. OM-MSCs-Exo have been reported to promote the proliferation and migration of human brain microvascular endothelial cells (HBMECs) [18], regulate microglial activation, reduce neuroinflammation, and enhance synaptic plasticity and neurogenesis via brain-derived neurotrophic factor (BDNF) [10]. However, the specific mechanism by which OM-MSCs-Exo alleviate neuroinflammation and cognitive impairment in AD remains unclear.

Microglia, the resident mononuclear phagocytes of the central nervous system, play crucial roles in both normal central nervous system (CNS) function and in response to Aβ accumulation. Their function is similar to that of blood macrophages, with phagocytic and inflammatory properties [20]. Microglial polarization is a critical determinant in the progression and regression of neurodegenerative diseases [21]. Microglia can be polarized into pro-inflammatory M1 or anti-inflammatory M2 phenotypes. Endogenous stimuli, such as reactive oxygen species, inflammatory factors, and accumulated Aβ, promote M1 polarization, contributing to neuronal damage. In contrast, M2 polarization mitigates neuroinflammation and Aβ toxicity in the AD brain [22, 23]. Cyanidin-3-O-glucoside (C3G) has been shown to reverse Aβ accumulation in AD models by shifting microglial polarization from M1 to M2 and upregulating the triggering receptor expressed on myeloid cells (TREM2) [24]. In cerebral ischemia–reperfusion injury, curcumin has been shown to alleviate neuroinflammation by promoting M2 polarization of microglial cells mediated by OM-MSCs [25]. Additionally, MSC-derived Exo have been reported to regulate microglial polarization [26]. However, the specific role of OM-MSCs-Exo in modulating microglial polarization in AD is still not well understood.

In this study, we aim to investigate whether OM-MSCs-Exo can regulate microglial polarization between M1 and M2 phenotypes through the secretion of specific proteins, thereby influencing neuroinflammation and cognitive impairment in AD. We will test this hypothesis using proteomics and in vivo and in vitro experiments. These findings may contribute to future breakthroughs in developing exosome-based therapies for AD.

Methods and Materials

Isolation and Culture of OM-MSCs

OM-MSCs were isolated from the nasal cavity of C57BL/6 mice as previously described [27]. Briefly, olfactory epithelial tissue was harvested, chopped into small pieces, and cultured in DMEM/F-12 medium (AW-MC025; Abiowell) supplemented with 15% fetal bovine serum (FBS, 10,099,141; Gibco) for 7 days. Non-adherent cells were removed, and the remaining cells were trypsinized and further expanded.

Characterization of OM-MSCs

For flow cytometric characterization of MSCs, MSCs were stained with fluorescently labeled monoclonal antibodies. The following fluorescently conjugated antibodies from eBioscience were used for flow cytometric analysis: anti-CD34-FITC (11–0341-82), anti-CD45-APC (17–0451-82), anti-CD79α-APC (MA5-50,653), anti-CD11b-PE (12–0112-82), anti-HLA-DR-PE (12–9956-42), anti-CD73-PE (12–0731-82), anti-CD90-PE (MA5-17,749), and anti-CD105-PE (12–1051-82). Corresponding isotype controls from the same supplier included Rat IgG2a kappa-FITC (clone eBR2a; 11–4321-80), Rat IgG2b kappa-APC (clone eB149/10H5; 17–4031-82), and Mouse IgG2b kappa-PE (clone eBMG2b; 12–4732-81). For osteogenic induction, OM-MSCs were cultured in osteogenic induction medium for 30 days and then stained with Alizarin Red S (C0148S; Beyotime) to assess mineralization. The osteogenic induction medium consisted of minimal essential medium supplemented with 0.1 μM dexamethasone (265,005; Sigma-Aldrich), 10 mM β-glycerophosphorus (G9422; Sigma-Aldrich), and 0.2 mM ascorbic acid (1,043,003; Sigma-Aldrich). For adipogenic differentiation, OM-MSCs were induced using a commercial adipogenic differentiation medium (A1067501; Thermo Fisher). After 30 days of culture, lipid droplets were stained with Oil Red O (OILR-10001; Cyagen) and visualized. Alcian blue staining was performed to assess chondrogenic differentiation. MO-MSCs were cultured in chondrogenic induction medium (HUXXC-90041; Cyagen) for 30 days. The resulting cell aggregates/micromasses were then fixed with 4% paraformaldehyde for 30 min at room temperature. Subsequently, the fixed samples were stained with Alcian blue staining solution (ALCB-10001; Cyagen) for 30 min in the dark to visualize sulfated proteoglycans, a key component of the cartilage extracellular matrix.

Isolation of OM-MSCs-Exo

OM-MSCs were cultured for 48 h in a culture medium with Exo-depleted FBS. After the supernatant was collected, we extracted the Exo using ultracentrifugation. Exo in the supernatant was purified by differential centrifugation to remove cells and debris, followed by ultracentrifugation at 100,000 × g for 60 min at 4 °C. After removal of the supernatant, the Exo pellet was washed in PBS and centrifuged again at 100,000 × g for 60 min at 4 °C before being resuspended in PBS and stored at − 80 °C.

Characterization of OM-MSCs-Exo

For transmission electron microscope (TEM) analysis, the OM-MSCs-Exo suspension was fixed in 4% paraformaldehyde for 60 min at 4 °C. Then, the Exo precipitate was applied to Formvar-coated grids and negatively stained with 3% aqueous phosphotungstic acid. TEM samples were imaged using a transmission electron microscope (Tecnai-12; Philips). For nanoparticle tracking analysis (NTA), the separated OM-MSCs-Exo was diluted with 1 × PBS buffer and then detected using Zeta view PMX 110. The size of OM-MSCs-Exo was measured via NTA 3.4 Build 3.4.4. The obtained OM-MSCs-Exo was subjected to Western blot (WB) analysis using antibodies against Galnexin, Tumor Susceptibility Gene 101 (TSG101), CD63, and CD81. In the WB section of this document, detailed steps and antibody information are provided.

LC–MS/MS Analysis for Astral-Data-Independent Acquisition

Appropriate amounts of SDS (sodium dodecyl sulfate) -DTT (dithiothreitol) -Tris (Tris (hydroxymethyl) aminomethane) (SDT) lysate were added to each cell sample and transferred to eppendorf tube (EP) tubes. Then, the cells were incubated with boiling water for 3 min, sonicated for 2 min, centrifuged at 16,000 × g for 20 min at 4 °C, and the supernatant collected. After the exosome samples were freeze dried, an appropriate amount of SDT lysate was added, and protein quantification was performed using the BCA Protein Assay Kit (P0012; BeyoTime). Proteins were reduced with 10 mM dithiothreitol (DTT) and cysteine at 37 °C for 30 min and then alkylated with 50 mM IAA in the dark at room temperature. Finally, proteins were diluted with 50 mM ammonium bicarbonate and digested overnight at 37 °C with trypsin (V5111; Promega) at a ratio of 50:1. The digested peptides were desalted using a C18 StageTip for further LC–mass spectrometry (MS) analysis. The concentration of polypeptides was determined by OD280 using a Nanodrop One device (840–317500; Thermo Fisher). LC–MS/MS was performed on an Orbitrap Astral mass spectrometer coupled with a Vanquish Neo UHPLC system (VN-S10-A-01; Thermo Fisher). Peptides from each sample were injected into a 50-cm Low-Load µPAC Neo HPLC Column (COL-LOLO050NEOB; Thermo Fisher) at a flow rate of 2.2 μL/min. The RP–HPLC mobile phase A was 0.1% formic acid in water, and B was 0.1% formic acid in 80% acetonitrile. Peptides were eluted over 8 min with a linear gradient of buffer B at 1.25 μL/min. The linear gradient was set as follows: 0–0.1 min, linear gradient from 4 to 6% buffer B. The eluted peptides were analyzed on an Orbitrap Astral mass spectrometer. The data-independent acquisition (DIA) method consisted of a survey scan from 380 to 980 m/z at a resolution of 240,000 with an AGC target of 500% and 5-ms injection time. The DIA MS/MS scans were acquired by Astral from 150 to 2000 m/z with a 2-m/z isolation window and with an AGC target of 500% and 3-ms injection time. The normalized collision energy was set to 25, and the cycle time was 0.6 s. By using the software DIA-NN (v1.9.2), all mass spectrometry data were combined, completing the database search and protein DIA quantitative analysis for the DIA mass spectrometry data. The database used was Uniprotkb-Mus musculus (Mice) [10090]−87434–2024-0725.fasta, which was sourced from the URL https://www.uniprot.org/taxonomy/10090.

Bioinformatics Analysis

In the significant difference analysis of protein expression between OM-MSCs and OM-MSCs-Exo, Student’s t-test was combined with the fold change (FC, the ratio of the average expression levels between the two groups) method to identify differentially expressed proteins (DEPs) (typically meeting the criteria of p-value < 0.05 and FC ≥ 1.5). A total of 8182 upregulated DEPs and 431 downregulated DEPs were obtained in OM-MSCs-Exo. The “Pheatmap” package (v1.0.12) was used for differential protein clustering analysis. The Gene Ontology (GO) delineated three aspects of biology: Cellular Component (CC), Biological Process (BP), and Molecular Function (MF) [28]. GO functional annotation and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis were used to annotate proteins. Protein–protein interaction (PPI) network analysis was performed using the STRING database (https://cn.string-db.org).

Culture of Cells

Mice-derived BV2 microglia (AW-CNM081) and HT-22 hippocampal neuron cells (AW-CNM116), along with human-derived HMC3 microglia cells (AW-CNH003) and SH-SY5Y neuronal cells (AW-CCH335), together with their corresponding culture media, were procured from Abiowell. STR identification reports were provided at the time of delivery. BV2 cells and HT-22 cells were grown in DMEM (AW-M008) containing 10% FBS and 1% penicillin–streptomycin (AWH0529a). HMC3 cells were cultured in complete MEM medium (iCell-0012; Icell), while SH-SY5Y cells were maintained in specialized MEM (iCell-h187-001b; Icell). All cells were cultured in an incubator at 37 °C, 5% CO₂, and saturated humidity. OM-MSCs were pretreated with or without 5 µM GW4869 (exosome release inhibitor, D1692; Sigma-Aldrich), and the conditioned medium (ECM) from OM-MSCs was collected. BV2 and HMC3 microglial cells, pretreated with Aβ_1–42 for 24 h, were co-cultured with ECM. [29]

Transfection and Grouping of Cells

The mice-targeted fibroblast growth factor receptor 1 (FGFR1) overexpression plasmid (oe-FGFR1), phospholipase C (PLC) γ1 small interfering RNA plasmid, and their control plasmids (oe-NC, si-NC) were purchased from Honorgene. After thawing on ice, 3 μg of oe-FGFR1 was added to a centrifuge tube containing 95 μL of special medium free of serum. Five microliters of Lip2000 was added to another centrifuge tube containing 95 μL of dedicated medium free of serum. The two tubes of solution were mixed and allowed to stand for 20 min at room temperature. The mixture was used to culture the transfected cells for 6 h and then replaced with a fresh medium. Other plasmid transfection procedures were the same.

Microglia cells (BV2 and HMC3) in the Control group were cultured under normal conditions. In the Aβ_1–42 group, microglia cells were treated with Aβ_1–42 (10 μM) for 24 h [30–32]. After the Aβ_1–42 treatment, microglia cells in the Aβ_1–42 + OM-MSCs-Exo group were treated with 40 μg/mL of OM-MSCs-Exo. Microglia cells in the Aβ_1–42 + OM-MSCs-Exo^oe−NC group and Aβ_1–42 + OM-MSCs-Exo^oe−FGFR1 group were treated with 40 μg/mL of OM-MSCs-Exo transfected overexpressing negative control (OM-MSCs-Exo^oe−NC) or OM-MSCs-Exo transfected overexpressing FGFR1 (OM-MSCs-Exo^oe−FGFR1) for 24 h, respectively [33]. In the Aβ_1–42 + OM-MSCs-Exo^oe−FGFR1 + si-NC group and the Aβ_1–42 + OM-MSCs-Exo^oe−FGFR1 + si-PLCγ1 group, microglia cells were transfected with si-NC and si-PLCγ1 plasmids, respectively, followed by Aβ_1–42 induction and treatment with OM-MSCs-Exo^oe−FGFR1. These exosomes (OM-MSCs-Exo, OM-MSCs-Exo^oe−NC, OM-MSCs-Exo^oe−FGFR1) were all isolated from the conditioned medium of transfected OM-MSCs and purified through standard ultracentrifugation steps, including washing. A Transwell co-culture system was utilized to co-culture HT-22 cells and SH-SY5Y cells from the respective experimental groups with correspondingly treated BV2 and HMC3 cells for 24 h.

Real-Time Quantitative Polymerase Chain Reaction

Microglia cells and mouse hippocampal tissues were added to Trizol for mRNA extraction. cDNA was synthesized with the use of an mRNA reverse transcription kit (CW2569; CWBIO). Real-time quantitative polymerase chain reaction (RT-qPCR) was performed using UltraSYBR Mixture (CW2601; CWBIO) on a fluorescence quantitative PCR system (QuantStudio1; ABI). The PCR cycle was 95 °C for 15 s and 60 °C for 30 s. The mRNA expression was assessed using the 2^−ΔΔCt method. β-Actin was used as an internal control. The primers used have been listed in Table S1.

Western Blot

After extracting proteins with RIPA (P0013B; Beyotime), the total protein concentration in the hippocampus of mice and microglia cells was determined by the BCA method. The proteins were separated by 10% sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) and transferred to nitrocellulose membranes. Membranes were blocked with 5% (w/v) skim milk powder for 90 min and incubated with primary antibodies overnight at 4 °C. Subsequently, the protein bands were incubated with secondary antibodies for 90 min. β-Actin was used as an internal control. Information on the antibodies used is listed in Table S2. Enhanced chemiluminescence (ECL) chemiluminescence solution was used to incubate the membrane for 1 min, and the gel imaging system (ChemiScope6100; CLINX) was used for imaging. The exposed image strips were analyzed with Quantity One professional grayscale analysis software (v4.6.8; BIO-RAD).

Co-immunoprecipitation Assay

The extracted cell proteins were randomly divided into Input, IgG, and IP groups. In the IgG and IP group centrifuge tubes, cell proteins were respectively incubated overnight at 4 °C with IgG (B900610; Proteintech) and FGFR1 primary antibodies. Then, 20 μL of protein A/G magnetic beads was added to each tube to capture the antibody–protein complexes. FGFR1 and FLCγ1 were analyzed by WB.

Immunofluorescence Staining

The microglia cell slides were fixed with 4% paraformaldehyde for 30 min. For mouse brain tissue paraffin sections, after dewaxing and hydration in xylene and ethanol, heat-induced antigen retrieval was performed using 0.01 M sodium citrate antigen retrieval buffer (pH 6.0, AWI0113a; Abiowell). They were then blocked with the prepared 5% PBS–BSA solution for 1 h. Subsequently, the cell or tissue slides were incubated overnight at 4 °C with the primary antibodies ionized calcium-binding adapter protein 1 (IBA1) (1:100, ab283346; Abcam), CD86 (1:50, AWA12696; Abiowell), and CD206 (1:50, AWA11319; Abiowell). After washing with PBS, the cell or tissue slides were incubated with fluorescein (FITC)-conjugated IgG (SA00003-11; Proteintech) for 90 min. DAPI was used to stain the nuclei. The slices were sealed with glycerol. In the dark, the cells were observed under a microscope (BA210T; Motic), and fluorescence images were collected.

Cellular Uptake of Exo

The OM-MSCs-Exo was mixed with 50 μL of PKH26 working solution (100 μM) in the dark and incubated for 30 min. A 1% BSA solution was used to terminate staining. Then, 1 mL of medium was added to OM-MSC-Exo and mixed. The mixture was added to BV2 cells. Cells were incubated at 37 °C for 6 h in an incubator. After DAPI (AWI0331a; Abiowell) staining, the cells were observed under a fluorescence microscope (BA410T; Motic). Microglia cells in the Control group were not treated with OM-MSC-Exo.

Cell-Counting-Kit-8 Assay

HT-22 cells or SH-SY5Y cells were seeded in 24-well plates (3 × 10 [4] per well). After the cells were grouped as described above, 300 μL of 10% Cell Counting Kit-8 (CCK8) (C0037; Beyotime) medium mix was added to the cells. After 4 h of incubation, the optical density values of HT-22 cells at 450 nm were measured using a multifunctional microplate reader (MB-530; HEALES).

Flow Cytometry Analysis

Following the aforementioned treatments, HT-22 or SH-SY5Y cells were trypsinized (without EDTA) and centrifuged at 2000 rpm for 5 min. The cell pellet was resuspended in 500 μL of binding buffer and then mixed with annexin V–APC and propidium iodide (PI) from the annexin V–APC/PI cell apoptosis detection kit (BL1934C; Biosharp). The cells were incubated in the dark for 10 min. The apoptosis rate was detected by flow cytometry.

Biochemical Tests

The cells were centrifuged at 1000 × g for 15 min at 4 °C to collect the supernatant. One hundred milligrams of mouse hippocampal tissue was ground into homogenates by adding 1 mL of PBS. Tissue homogenates were centrifuged at 5000 × g for 5 min at 4 °C to obtain the supernatant. The supernatant was detected immediately. According to the instruction manuals of the mouse interleukin (IL)−1β (CSB-E08054m; Cusabio), tumor necrosis factor (TNF)-α (CSB-E04741m; Cusabio), IL-6 (CSB-E04639m; Cusabio), and Aβ_1–42 (CSB-E10787m; Cusabio) detection kits, the corresponding molecular levels in cells or tissues were measured.

Animal Experiments

Male C57BL/6J mice (6 weeks of age) weighing 18–22 g were purchased from Hunan SJA Laboratory Animal Co. Mice were acclimatized for 1 week prior to the experiment with a 12-h/12-h light–dark cycle. Meanwhile, Aβ_1–42 (A494432; Aladdin) was dissolved in sterile saline at a concentration of 5 μg/μL. The mice were randomly assigned to five groups, with six mice in each group. Mice were anesthetized by intraperitoneal injection of 40 mg/kg sodium pentobarbital and immobilized on an operating table. Aβ_1–42 (5 μg/side) was injected into the bilateral hippocampus of mice in the Aβ_1–42 group (anteroposterior position − 2.0 mm, medial–lateral position ± 1.8 mm, dorsoventral distance 2 mm from Bregma) through a stereotaxic instrument at 0.2 μL/min [34]. Mice in the Sham group were injected with the same volume of normal saline. Seven days after modeling, 100 μg of purified OM-MSCs-Exo, OM-MSCs-Exo^oe−NC, and OM-MSCs-Exo^oe−FGFR1 were each dissolved in 100 μL of PBS and injected via the tail vein once a week into the Aβ_1–42 + OM-MSCs-Exo group, OM-MSCs-Exo^oe−NC group, and OM-MSCs-Exo^oe−FGFR1 group of mice, respectively, for eight consecutive weeks [35]. Behavioral tests were conducted 1 week after the final intervention. Subsequently, all mice were anesthetized and perfused with physiological saline solution through the heart, and the brains were harvested. Each half of the brain was rapidly frozen in liquid nitrogen and stored at − 80 °C for protein collection, while the other half of the hippocampal tissue was fixed, dehydrated, and sectioned into 4-μm paraffin slices for immunostaining.

Morris Water Maze Tests

Mice were placed in a circular pool (diameter 1.2 m) filled with water (25 ± 1 °C) and visual cues were placed around the pool. The Morris water maze (MWM) test consisted of a visible test (day 0), a hidden platform test (days 1–5), and a probe test (day 6) [36]. Mice were released into the pool 6 times a day during visible and hidden platform testing. The number of platform entries and the latency of the first entry into the target quadrant were recorded. During the probe trial, the platform is removed, allowing the mice to swim freely for 60 s. Swimming distance and swimming time were recorded.

TdT-Mediated dUTP Nick-End Labeling Staining

Paraffin sections of mouse brain tissue were baked, dried, and placed in xylene. The sections were then treated in 100%, 100%, 95%, 85%, and 75% ethanol for 5 min in sequence. Then, 100 μL of Proteinase K working solution was added to each sample, and the reaction was carried out at 37 °C for 20 min. According to the manufacturer’s instructions, sections were stained with a TdT-mediated dUTP nick-end labeling (TUNEL) staining kit (40306ES50; YEASEN) and a DAPI working solution under dark conditions. The slices were sealed with glycerol. Fluorescence microscopy was used to acquire images.

Histochemical Staining

For hematoxylin and eosin (HE) staining, the mouse brain tissue paraffin sections were dewaxed and hydrated, followed by staining with hematoxylin (AWI0001a; Abiowell). PBS was used for bluing. After eosin staining, the sections underwent gradient alcohol dehydration (95–100%). Following mounting with neutral gum, the slides were observed under a microscope (BA210T; Motic).

For Nissl staining, after dewaxing and hydration of the mouse brain tissue paraffin sections, they were stained with Nissl stain (AWI0501a; Abiowell) for 30 s. Following destaining, differentiation in 1% glacial acetic acid was performed until the background appeared colorless under the microscope, while the cells and Nissl bodies exhibited clear outlines. Finally, the sections were observed under the microscope.

Statistical Analysis

All data are presented as mean ± standard deviation (SD). Statistical comparisons of the results were conducted using Prism software (v10.2). After passing the normality test, an unpaired two-tailed Student’s t-test was employed for analyzing the data between the two groups. Data among multiple groups were analyzed by one-way analysis of variance (ANOVA), followed by Tukey’s post hoc test. R software (v4.4.2) was utilized for bioinformatics analysis. A significance level of p < 0.05 was considered statistically significant.

Results

OM-MSCs-Exo Was Successfully Prepared

We isolated OM-MSCs from mouse nasal olfactory epithelial tissue and analyzed the surface markers of the cells by flow cytometry. The results showed that the isolated cells had a CD34-positive rate of 1.18%, a CD45-positive rate of 0.17%, a CD11b-positive rate of 1.93%, a CD79α-positive rate of 1.21%, an HLA-DR-positive rate of 0.48%, a CD73-positive rate of 99.21%, a CD90-positive rate of 98.32%, and a CD105-positive rate of 95.10% (Fig. 1A). Under microscopic examination, OM-MSCs exhibited adherent growth with a spindle-shaped or elongated fibroblast-like morphology (Fig. 1B). The staining results with Alizarin Red S, Oil Red O, and Alcian blue demonstrated that, after 1 month of culture under respective induction conditions, OM‑MSCs successfully differentiated into osteoblasts, adipocytes, and chondrocytes, confirming their trilineage differentiation potential (Fig. 1C). These results collectively indicated that OM-MSCs exhibited typical characteristics of MSCs and were successfully purified.

Fig. 1.

OM-MSCs and OM-MSCs-Exo were successfully purified. A The immunophenotypes of OM-MSCs were detected by flow cytometry, including CD34, CD45, CD14/CD11b, CD79α/CD19, HLA-DR, CD73, CD90, and CD105. B Morphological images of OM-MSCs were captured under a microscope. C After 30 days of induction toward osteogenic, adipogenic, and chondrogenic differentiation, OM-MSCs were subjected to Alizarin Red S, Oil Red O, and Alcian blue staining, respectively. D TEM analysis of OM-MSCs-Exo. E NTA of OM-MSCs-Exo. F WB analysis of Calnexin, TSG101, CD63, and CD81

The purified OM-MSCs-Exo exhibited a typical spherical shape surrounded by a double membrane, and the NTA indicated an average diameter of 153.1 nm, consistent with the size of exosomes (Fig. 1D, E). WB analysis revealed that the isolated OM-MSCs-Exo exhibited characteristic exosomal markers, including TSG101, CD63, and CD81, while lacking the endoplasmic reticulum marker Calnexin. In contrast, OM-MSCs exhibited a positive expression of Calnexin, while TSG101, CD63, and CD81 were only minimally expressed (Fig. 1F). These findings confirmed the successful isolation and purification of OM-MSCs-Exo.

OM-MSCs-Exo Promote M2 Polarization of Microglia In Vitro

The effect of OM-MSCs-Exo on microglial polarization was examined in vitro. The immunofluorescence (IF) staining results showed that compared to the Control group, Aβ_1–42 induction increased the proportions of both IBA1⁺CD206⁺ and IBA1⁺CD86⁺ microglial cells. Relative to the Aβ_1–42 group, the Aβ_1–42 + OM-MSCs ECM group exhibited a further increase in IBA1⁺CD206⁺ microglia, while the proportion of IBA1⁺CD86⁺ cells decreased. Inhibition of exosome secretion partially reversed the ECM-mediated effects on microglial polarization (Fig. 2A). Furthermore, treatment with Aβ_1–42 upregulated the expression of both M1 polarization markers (iNOS, IL-1β, IL-18, TNF-α) and M2 polarization markers (Arg-1, IL-10, TGF-β) in microglia. Co-culture with OM-MSCs ECM enhanced the levels of Aβ_1–42-induced M2 markers while reducing the levels of M1-related factors. The effects of OM-MSCs’ ECM were reversed by intervention with the exosome release inhibitor (Fig. 2B). The data imply that OM-MSCs may exert their pro-M2 polarizing effect on microglia through the exosomes.

Fig. 2.

OM-MSCs-Exo polarizes microglia toward the M2 phenotype. A IF staining was performed to determine the co-localization positivity rate of the microglial marker IBA1 with CD86 and CD206, respectively. B The mRNA level of iNOS, IL-1β, IL-18, TNF-α, Arg1, IL-10, and TGF-β. The normality of data was assessed using the Shapiro–Wilk test. Subsequently, comparisons among multiple groups were performed using one-way ANOVA, with Tukey’s test applied for post hoc analysis

Proteomic Analysis of DEPs in OM-MSCs-Exo

We performed a proteomic analysis to identify DEPs between OM-MSCs-Exo and OM-MSCs and generated a volcano plot and heatmap representations (Fig. 3A, B). Based on the protein expression levels in OM-MSCs and OM-MSCs-Exo, we identified the top five DEPs that were significantly enriched in OM-MSCs-Exo compared to OM-MSCs: Serpinc1, FEN1, CNOT1, FGFR1, and ITIH4. The heatmap showed that compared to OM-MSCs, the protein levels of Serpinc1, FEN1, CNOT1, FGFR1, and ITIH4 were significantly upregulated in OM-MSCs-Exo. According to the GO functional enrichment analysis, these DEPs are primarily enriched in the Biological Process category, including the RNA metabolic process and nucleic acid metabolic process pathways; the Cellular Component category, including intracellular membrane–bounded organelle and intracellular anatomical structure components; and the Molecular Function category, nucleic acid binding, RNA binding, and organic cyclic compound binding (Fig. 3C). KEGG analysis revealed that the DEPs in OM-MSCs-Exo were significantly enriched in pathways such as autophagy—animal, adherens junction, and AD (Fig. 3D). We then performed WB analysis to detect the expression of the top 5 DEPs in OM-MSCs and OM-MSCs-Exo. Compared to OM-MSCs, the expression of Serpinc1, FEN1, CNOT1, FGFR1, and ITIH4 increased in OM-MSCs-Exo, with FGFR1 showing the most significant difference (Fig. 3E). Relative to the Sham group mice, the levels of Serpinc1, FEN1, CNOT1, FGFR1, and ITIH4 proteins decreased in the hippocampal tissue of Aβ_1–42 group mice, with FGFR1 showing the most significant decrease (Fig. 3F). Thus, FGFR1, showing the most pronounced downregulation, was chosen for subsequent study.

Fig. 3.

Differentially expressed proteins between OM-MSCs and OM-MSCs-Exo were screened by proteomic analysis. A DEPs were identified using a volcano map. B Heatmap clustering analysis of DEPs. C Based on GO enrichment analysis of all DEPs, the top 10 pathways were selected in BP, CC, and MF, respectively. D KEGG enrichment analysis based on all DEPs was performed to select the top 20 pathways. E WB analysis of Serpinc1, FEN1, CNOT1, FGFR1, and ITIH4 in OM-MSCs and OM-MSCs-Exo (n = 3). F WB analysis of Serpinc1, FEN1, CNOT1, FGFR1, and ITIH4 in mice hippocampal tissue (n = 5). ***p < 0.001. The normality of the data was assessed using the Shapiro–Wilk test. For comparisons between two groups, an unpaired two-tailed Student’s t-test was used

Delivery of FGFR1 by OM-MSCs-Exo Induced M2 Polarization of Microglia In Vitro

IBA1 was used as a specific marker for microglial cells [37]. The mouse BV2 cells and human HMC3 cells purchased by us showed positive staining for IBA1 (Fig. 4A). After labeling OM-MSCs-Exo with PKH26, they were co-cultured with microglial cells. Compared to the Control group, microglia incubated with PKH26-labeled OM-MSC-Exos exhibited positive PKH26 IF staining (Fig. 4B). This result indicated that OM-MSCs-Exo were internalized by microglial cells. Next, we further investigated through in vitro experiments whether OM-MSCs-Exo influenced the polarization of microglial cells by secreting FGFR1. After adding Aβ_1–42, the level of p-FGFR1 and FGFR1 proteins decreased in microglial cells. Compared to the Aβ_1–42 group, OM-MSCs-Exo increased the level of p-FGFR1 and FGFR1 in microglial cells (Fig. 4C). After transfection with the oe-FGFR1 plasmid, FGFR1 protein expression was increased in OM-MSCs-Exo, whereas its mRNA level showed no significant change (Fig. 4D). Compared to the Aβ_1–42 + OM-MSCs-Exo^oe−NC group, the Aβ_1–42 + OM-MSCs-Exo^oe−FGFR1 group showed a further upregulation of FGFR1 expression in microglial cells. However, FGFR1 mRNA levels showed no significant difference between the two groups (Fig. 4E, F). Aβ_1–42 induction resulted in an increase in the M1 polarization marker CD86 in microglial cells, which was reversed by OM-MSCs-Exo treatment. Furthermore, OM-MSCs-Exo^oe−FGFR1 further enhanced the inhibitory effect of OM-MSCs-Exo on CD86 expression induced by Aβ_1–42 in microglial cells. Conversely, the M2 polarization marker CD206 showed an opposite pattern (Fig. 4G). Aβ_1–42 promoted the secretion of M1 polarization–related factors (inducible nitric oxide synthase (iNOS), IL-1β, IL-18, and TNF-α) by microglial cells and suppressed the production of M2 polarization–related factors (Arginase 1 (Arg1), IL-10, and transforming growth factor (TGF)-β). Conversely, OM-MSCs-Exo inhibited the secretion of M1 polarization–related factors induced by Aβ_1–42 in microglial cells while increasing the levels of M2 polarization–related factors. Compared with the Aβ_1–42 + OM-MSCs-Exo^oe−NC group, OM-MSCs-Exo^oe−FGFR1 further enhanced the effect of OM-MSCs-Exo (Fig. 4H). The results mentioned above indicated that OM-MSCs-Exo promoted microglial cell polarization toward an M2 phenotype through FGFR1.

Fig. 4.

OM-MSCs-Exo-delivered FGFR1 induced the M2 polarization in microglial cells. A IF staining of IBA1. B PKH26-labeled Exo was taken up by microglial cells. C The p-FGFR1 and FGFR1 expression of microglial cells was analyzed by WB. D The FGFR1 level of OM-MSCs-Exo was assessed by RT-qPCR and WB. E The FGFR1 expression was analyzed by RT-qPCR in microglial cells. F The FGFR1 expression was analyzed by WB in microglial cells. G IF staining of IBA1, CD86, and CD206. H The level of iNOS, IL-1β, IL-18, TNF-α, Arg1, IL-10, and TGF-β (n = 3). *p < 0.05, **p < 0.01, ***p < 0.001. Normality was confirmed using the Shapiro–Wilk test. Thereafter, data were analyzed with a one-way ANOVA (followed by Tukey’s post hoc test) for multiple-group comparisons or with an unpaired two-tailed t-test for comparisons between two groups

OM-MSCs-Exo Transferred FGFR1 to Induce M2 Polarization of Microglia, Thereby Suppressing Neuronal Inflammation Response

Under the condition of excluding the influence of microglial co-culture, OM-MSCs-Exo enhanced the microglial activity of BV2 and HMC3 inhibited by Aβ_1–42. OM-MSCs-Exo^oe−FGFR1 further enhanced this effect (Supplementary Fig. S1). To further verify whether OM-MSCs-Exo inhibited neuronal inflammation by inducing M2 polarization of microglia through the delivery of FGFR1, the BV2 and HMC3 microglial cells from the aforementioned groups were co-cultured with HT-22 or SH-SY5Y neuronal cells, respectively. The viability of neuronal cells co-cultured with microglial cells induced by Aβ_1–42 was decreased, and the apoptosis rate was increased compared to the Co-control group. In the Co-Aβ_1–42 + OM-MSCs-Exo group, the effects of Aβ_1–42 were reversed. In comparison to the Co-Aβ_1–42 + OM-MSCs-Exo^oe−NC group, the microglial cells treated with OM-MSCs-Exo^oe−FGFR1 enhanced the activity of neuronal cells and reduced the apoptosis of neuronal cells (Fig. 5A, B). Aβ_1–42 induction of microglial cells increased the expression of IL-1β, TNF-α, and IL-6 in HT-22 cells. IL-1β, TNF-α, and IL-6 expression in neuronal cells was decreased in the Co-Aβ_1–42 + OM-MSCs-Exo group relative to the Co-Aβ_1–42 group. Compared to the Co-Aβ_1–42 + OM-MSCs-Exo^oe−NC group, the production of inflammatory factors in neuronal cells was further suppressed by the microglial cells treated with Aβ_1–42 + OM-MSCs-Exo^oe−FGFR1 (Fig. 5C). Combining the results from Figs. 3 and 4, OM-MSCs-Exo might induce M2 polarization of microglial cells through the secretion of FGFR1, thereby suppressing inflammation in neuronal cells.

Fig. 5.

OM-MSCs-Exo induced M2-polarized microglial cells through FGFR1 delivery, resulting in attenuated neuronal inflammation. A CCK8 assay in HT-22 and SH-SY5Y cells. B The apoptosis rate of HT-22 and SH-SY5Y cells was analyzed by flow cytometry. C IL-1β, TNF-α, and IL-6 levels of HT-22 and SH-SY5Y cells. The HT-22 cells in the Co-control group, Co-Aβ_1–42 group, Co-Aβ_1–42 + OM-MSCs-Exo group, Co-Aβ_1–42 + OM-MSCs-Exo^oe−NC group, and Co-Aβ_1–42 + OM-MSCs-Exo^oe−FGFR1 group were co-cultured with the corresponding BV2 cells for 24 h. The SH-SY5Y cells in the Co-control group, Co-Aβ_1–42 group, Co-Aβ_1–42 + OM-MSCs-Exo group, Co-Aβ_1–42 + OM-MSCs-Exo^oe−NC group, and Co-Aβ_1–42 + OM-MSCs-Exo^oe−FGFR1 group were co-cultured with the corresponding HMC3 cells for 24 h (n = 3). *p < 0.05, **p < 0.01, ***p < 0.001. Normality was confirmed using the Shapiro–Wilk test. Thereafter, data were analyzed with a one-way ANOVA (followed by Tukey’s post hoc test) for multiple-group comparisons

OM-MSCs-Exo Transferred FGFR1 to Induce M2 Polarization of Microglial Cells In Vitro through Interacting with PLCγ1

To further elucidate the mechanism by which FGFR1 induced M2 polarization of microglial cells, we obtained a PPI network of FGFR1 (Fig. 6A). Among the candidate interactors identified, we focused on phospholipase C gamma 1 (PLCγ1) based on its established role in inflammatory signaling and its relevance to the observed phenotype. Notably, PLCγ1 levels were downregulated in the hippocampal tissue of Aβ_1–42-treated mice compared to the Sham group (Fig. 6B), suggesting its potential involvement in AD-related neuroinflammation. The invariant tyrosine of FGFR (Y766 in FGFR1) in the C terminus of FGFR is phosphorylated, creating a binding site for the SH2 structural domain of PLCγ, which is necessary for the phosphorylation and activation of PLCγ [38, 39]. Aβ_1–42 decreased the levels of PLCγ1 in microglial cells. OM-MSCs-Exo increased the expression of PLCγ1 in Aβ_1–42-induced BV2 cells. Relative to the OM-MSCs-Exo^oe−NC, OM-MSCs-Exo^oe−FGFR1 exhibited a more significant upregulation effect on PLCγ1 expression in Aβ_1–42-induced microglial cells (Fig. 6C). The co-immunoprecipitation assay confirmed the interaction between FGFR1 and PLCγ1 (Fig. 6D).

Fig. 6.

FGFR1 secreted by OM-MSCs-Exo induced the polarization of microglial cells to the M2 type through interaction with PLCγ1. A PPI analysis of FGFR1. B PLCγ1 protein levels in mice hippocampus. C PLCγ1 protein levels in BV2 cells and HMC3 cells. D Protein interaction between FGFR1 and PLCγ1 was observed. E PLCγ1 levels. F PLCγ1 mRNA and protein levels. G Levels of p-NF-κB/NF-κB. H IF staining of IBA1, CD86, and CD206. I M1 polarization–related factor and M2 polarization–related factor levels in microglial cells (n = 3). *p < 0.05, **p < 0.01, ***p < 0.001. Normality was confirmed using the Shapiro–Wilk test. Thereafter, data were analyzed with a one-way ANOVA (followed by Tukey’s post hoc test) for multiple-group comparisons or with an unpaired two-tailed t-test for comparisons between two groups

Transfection of si-PLCγ1 (#1 and #2) significantly suppressed the expression of PLCγ1 in microglial cells compared to the si-NC plasmid (Fig. 6E). The si-PLCγ1#2 plasmid was used for subsequent studies. Downregulation of PLCγ1 reversed the upregulation effect of OM-MSCs-Exo^oe−FGFR1 on PLCγ1 (Fig. 6F). Compared to the Aβ_1–42 + OM-MSCs-Exo^oe−NC group, the phosphorylated nuclear factor kappa B (p-NF-κB)/NF-κB ratio in microglia was significantly reduced by OM-MSCs-Exo^oe−FGFR1. This effect was reversed upon PLCγ1 knockdown (Fig. 6G). The OM-MSCs-Exo^oe−FGFR1 treatment resulted in a downregulation of CD86 and an upregulation of CD206 expression in BV2 cells. Transfection of PLCγ1 reversed this effect, as shown in Fig. 6H. In addition, OM-MSCs-Exo^oe−FGFR1 inhibited the production of iNOS, IL-1β, IL-18, and TNF-α in BV2 cells, while promoting the release of Arg-1, IL-10, and TGF-β. Knockdown of PLCγ1 reversed the effects of OM-MSCs-Exo^oe−FGFR1 (Fig. 6I). This indicated that the FGFR1 delivered by OM-MSCs-Exo can promote M2 polarization of microglial cells through PLCγ1.

OM-MSCs-Exo Delivered FGFR1 to Inhibit Neuronal Inflammation In Vitro by Interacting with PLCγ1

To further confirm whether OM-MSCs-Exos influenced neuronal inflammation through the FGFR1/PLCγ1 signaling axis in BV2 cells, we measured the levels of inflammatory factors in HT-22 cells co-cultured with BV2 cells from each group. Compared with the Co-Aβ_1–42 + OM-MSCs-Exo^oe−NC group, OM-MSCs-Exo^oe−FGFR1 increased the cell viability, reduced neuronal apoptosis, and inhibited the production of IL-1β, TNF-α, and IL-6 in neuronal cells. Compared with the Co-Aβ_1–42 + OM-MSCs-Exo^oe−FGFR1 + si-NC group, after knocking down PLCγ1 in BV2 cells, neuronal cell activity was weakened, apoptosis rate was increased, and the levels of IL-1β, TNF-α, and IL-6 were increased (Fig. 7A–C). Based on the results from Figs. 5 and 6, it was possible that OM-MSCs-Exo promoted M2 polarization of BV2 cells through the FGFR1/PLCγ1 axis, thereby suppressing neuronal inflammation.

Fig. 7.

OM-MSCs-Exo delivered FGFR1 to interact with PLCγ1 in microglia, suppressing the inflammatory response of co-cultured HT-22 and SH-SY5Y cells. A CCK8 assay in HT-22 and SH-SY5Y cells. B The apoptosis rate of neurons cells was analyzed by flow cytometry. C IL-1β, TNF-α, and IL-6 levels of HT-22 and SH-SY5Y cells. The HT-22 cells in the Co-Aβ_1–42 + OM-MSCs-Exo^oe−NC group, Co-Aβ_1–42 + OM-MSCs-Exo^oe−FGFR1 group, Co-Aβ_1–42 + OM-MSCs-Exo^oe−FGFR1 + si-NC group, and Co-Aβ_1–42 + OM-MSCs-Exo^oe−FGFR1 + si-PLCγ1 group were co-cultured with the corresponding BV2 cells for 24 h. The SH-SY5Y cells in the Co-Aβ_1–42 + OM-MSCs-Exo^oe−NC group, Co-Aβ_1–42 + OM-MSCs-Exo^oe−FGFR1 group, Co-Aβ_1–42 + OM-MSCs-Exo^oe−FGFR1 + si-NC group, and Co-Aβ_1–42 + OM-MSCs-Exo^oe−FGFR1 + si-PLCγ1 group were co-cultured with the corresponding HMC3 cells for 24 h (n = 3). *p < 0.05, **p < 0.01, ***p < 0.001. Normality was confirmed using the Shapiro–Wilk test. Thereafter, data were analyzed with a one-way ANOVA (followed by Tukey’s post hoc test) for multiple-group comparisons

OM-MSCs-Exo-Mediated Delivery of FGFR1 Alleviated Neuroinflammation and Cognitive Impairment in AD Mice

In vivo, we further validated the in vitro results. After intracerebroventricular injection of Aβ_1–42, mice showed decreased swimming distance and time during the MWM test, reduced the number of platform entries, and increased the latency to first entry into the target quadrant. Compared to the Aβ_1–42 group, intravenous injection of OM-MSCs-Exo reversed the outcomes induced by Aβ_1–42. Furthermore, OM-MSCs-Exo^oe−FGFR1 enhanced the effects of OM-MSCs-Exo (Fig. 8A). In the Sham group, the neurons in the hippocampal tissue of mice were densely arranged with normal structure. In contrast, in the AD mice induced by Aβ_1–42, the neurons in the hippocampal tissue were significantly decreased, loosely arranged, and exhibited nuclear shrinkage. OM-MSCs-Exo treatment alleviated neuron damage induced by Aβ_1–42, with partial restoration of neuronal nuclear morphology. Comparatively, OM-MSCs-Exo^oe−FGFR1 exhibited a more pronounced alleviation of neuron damage induced by Aβ_1–42 than OM-MSCs-Exo^oe−NC (Fig. 8B, C). Aβ_1–42 induced neuronal apoptosis in the hippocampus of mice, which was reversed by OM-MSCs-Exo. Compared with the Aβ_1–42 + OM-MSCs-Exo^oe−NC group, OM-MSCs-Exo^oe−FGFR1 inhibited neuronal apoptosis in AD mice (Fig. 8D, E). Compared to the Sham group, the levels of IL-1β, TNF-α, and IL-6 were elevated in the hippocampal tissue of AD mice, accompanied by increased Aβ deposition and p-Tau/Tau ratio. Injection of OM-MSCs-Exo reversed the aforementioned outcomes. Relative to the intervention with OM-MSCs-Exo^oe−NC, the overexpression of FGFR1 in OM-MSCs-Exo exhibited a more pronounced suppression of inflammatory factors, Aβ deposition, p-Tau/Tau ratio, and Aβ_1–42 levels (Fig. 8F–H). Aβ_1–42 induction downregulated the expression of FGFR1 and PLCγ1 in the hippocampal tissue of mice, while injection of OM-MSCs-Exo increased the expression of FGFR1 and PLCγ1 in AD mice. Compared with the Aβ_1–42 + OM-MSCs-Exo^oe−NC group, the expression of FGFR1 and PLCγ1 in the hippocampus of the Aβ_1–42 + OM-MSCs-Exo^oe−FGFR1 group was further increased (Fig. 8I). Meanwhile, Aβ_1–42 induced an increase in the level of p-NF-κB/NF-κB in the hippocampal tissue of mice. OM-MSCs-Exo reversed the effect of Aβ_1–42. After OM-MSCs-Exo overexpressed FGFR1, the p-NF-κB/NF-κB signaling pathway was further inhibited (Fig. 8J). Based on the above results, OM-MSCs-Exo could reduce cognitive function and neuroinflammation in AD mice. This function of OM-MSCs-Exo may be achieved through the signal transduction between FGFR1 secreted by OM-MSCs-Exo and PLCγ1 of microglia.

Fig. 8.

OM-MSCs-Exo alleviated cognitive impairment and neuroinflammation in AD mice through FGFR1. A Swimming distance, swimming time, number of platform arrivals, and latency to first entry (n = 6). B The hippocampal tissues of mice were stained with HE. C Nissl staining was performed in the hippocampus of mice. D TUNEL assay. E Data plot of the TUNEL assay. F Levels of IL-1β, TNF-α, and IL-6 in mice hippocampus. G WB analysis of Aβ, p-Tau/Tau in mice hippocampus. H Aβ_1–42 levels were detected. I FGFR1 and PLCγ1 levels were measured. J Levels of p-NF-κB/NF-κB. K, L IF staining of CD86 and CD206 in mice hippocampus. M Levels of microglia M1 and M2 polarization–related factors in mice hippocampus (n = 5). *p < 0.05, **p < 0.01, ***p < 0.001. Normality was confirmed using the Shapiro–Wilk test. Thereafter, data were analyzed with a one-way ANOVA (followed by Tukey’s post hoc test) for multiple-group comparisons

In terms of microglial polarization, the expression of the M1-type microglial marker CD86 was upregulated, and the M2-type microglial marker CD206 was downregulated in the hippocampal tissue of mice induced by Aβ_1–42. OM-MSCs-Exo reduced the levels of CD86 in the hippocampal tissue of AD mice while concurrently upregulating the expression of CD206. Moreover, OM-MSCs-Exo^oe−FGFR1 enhanced the effects of OM-MSCs-Exo (Fig. 8K, L). In comparison to the Sham group, levels of M1 polarization–associated factors (iNOS, IL-1β, IL-18, and TNF-α) were increased in the hippocampal tissue of mice in the Aβ_1–42 group, while levels of M2 polarization–associated factors (Arg1, IL-10, and TGF-β) were decreased. OM-MSCs-Exo reversed the effects of Aβ_1–42, and OM-MSCs-Exo^oe−FGFR1 further enhanced the effects of OM-MSCs-Exo (Fig. 8M). Combining the cellular experiments, OM-MSCs-Exo may promote M2 polarization of microglial cells in AD mice through the secretion of FGFR1, thereby alleviating cognitive impairments and neuroinflammation in AD mice.

Discussion

Neuroinflammation has been proposed as the third major pathological driver of AD after Aβ deposition and neurofibrillary tangles [40]. Recent studies in the scientific community suggested that extracellular vesicles derived from stem cells held great promise as a treatment modality for CNS diseases [41]. In this study, we found that OM-MSCs-Exo could promote microglial M2 polarization and reduce neuroinflammation in AD mice through the secretion of FGFR1 protein.

Cone and colleagues revealed that extracellular vesicles derived from MSCs could improve cognitive impairments in a preclinical AD mouse model [42]. The intranasal delivery of MSCs and their conditioned medium (including MSCs-Exo) could modulate the differentiation of microglial cells by inhibiting the polarization towards the pro-inflammatory M1 phenotype [43]. Consistent with reports that MSC-derived vesicles can improve cognitive deficits in AD models, we found that OM-MSCs-Exo administered via tail vein injection reduced Aβ deposition, decreased the p-Tau/Tau ratio, and improved cognitive function in AD mice. It was reported that OM-MSCs alleviate neuronal apoptosis induced by brain ischemia/reperfusion injury by modulating microglial polarization [25]. In vitro, OM-MSCs-Exo reversed Aβ_1–42-induced M1 polarization and enhanced the M2 phenotype in BV2 and HMC3 microglia. This shift was associated with reduced neuronal apoptosis in co-culture models. These results align with previous studies showing that OM-MSCs can modulate microglial polarization and promote Aβ clearance. Inhibition of exosome secretion using GW4869 abolished the pro-M2 effects, confirming the exosome-dependent mechanism. This indicates that OM-MSCs exert a protective effect by promoting M2 polarization of microglia through exosomes.

Proteomic analysis of OM-MSCs-Exo identified FGFR1 as one of the most upregulated proteins. Notably, the upregulation observed at the protein level was not accompanied by a corresponding change in FGFR1 mRNA, suggesting a post-transcriptional regulatory mechanism. Recent studies reported that FGFR1 knockdown in the mouse brain resulted in reduced cognitive function, impaired synaptic plasticity, and upregulated neuroinflammation [44]. After overexpressing FGFR1 in OM-MSCs-Exo, the M1 polarization of microglia cells and neuronal inflammatory responses in the AD model were further suppressed, while the M2 polarization showed the opposite result. The therapeutic effects of OM-MSCs-Exo on cognitive decline and neuroinflammation in AD mice were also enhanced by OM-MSCs-Exo^oe−FGFR1. Therefore, these findings suggest that OM-MSCs-Exo may alleviate neuroinflammation in AD mice by regulating microglial activation through FGFR1 protein secretion.

We further investigated the downstream signaling of FGFR1. Through PPI analysis and experimental validation, we identified PLCγ1 as a key effector. Activating the PLCγ1 signaling pathway suppressed the expression levels of neurotoxic pro-inflammatory mediators and cytokines, thereby significantly attenuating the overactivation of pro-inflammatory microglial cells (M1) [45]. Specifically, our data indicate that FGFR1 delivered by OM-MSCs-Exo engages PLCγ1 in microglia, thereby promoting their polarization toward the M2 phenotype and reducing neuronal damage in co-culture models. This finding is supported by both in vitro and in vivo evidence. Additionally, PLCγ1 can regulate the NF-κB pathway either through PKC or direct interaction. [45, 46] Overexpression of FGFR1 in OM-MSCs-Exo significantly reduced the p-NF-κB/NF-κB ratio in microglia, an effect that was abolished by PLCγ1 knockdown. The in vivo findings closely aligned with the in vitro observations.

Based on bioinformatics and literature evidence, the key interactors of FGFR1 can be categorized into upstream ligands (e.g., FGF2, FGF4, responsible for receptor activation), downstream effectors (with PLCγ1 as the core, mediating PKC/calcium signaling), and regulatory proteins (e.g., the co-receptor Klotho Beta (KLB), which enhances ligand-specific responses). Among these, the FGFR1/PLCγ1 axis serves as the central hub of this study. The activation of this axis depends on the binding of upstream ligands to FGFR1. Once activated, PLCγ1 converts the signal into second messengers, directly driving downstream cellular responses. Meanwhile, other interactors, such as KLB, help define the specific biological context of the signaling pathway. As a crucial member of the phospholipase C family, PLCγ1 plays a central role in multiple signaling pathways. Upon activation, it hydrolyzes phosphatidylinositol 4,5-bisphosphate (PIP2) to generate inositol trisphosphate (IP3) and DAG, thereby regulating downstream molecules and impacting processes such as cell proliferation, differentiation, migration, and apoptosis [47]. PLCγ1 activates protein kinase C (PKC) by generating diacylglycerol (DAG) to regulate various cellular functions [48]. Therefore, the FGFR1/PLCγ1 axis integrates information from a broad interaction network, forming the core signaling pathway that connects receptor activation to key phenotypic and functional changes in cells.

This study has several limitations. We characterized only the proteome of OM-MSC-Exo, without analyzing its RNA or lipid cargo, which may be important for understanding its full mechanism of action. Furthermore, the acute Aβ model used here induces inflammation but does not replicate the chronic progression or tau pathology of human AD. In addition, the in vivo distribution and targeting properties of the administered exosomes require further investigation. Mechanistically, the current focus of this study is on preliminarily identifying direct interacting partners of FGFR1 through proteomics and establishing a validation framework. We have not yet integrated large-scale omics data to dynamically and systematically resolve its regulatory network, which represents a limitation of the present work.

Although the role of the FGFR1/PLCγ1 pathway in neuroimmune regulation has been reported, the innovation of this work lies in being the first to reveal the key role of exosomes derived from OM-MSCs as a unique delivery system for this pathway in an AD-related neuroinflammation model. This provides a new theoretical basis for exosome-based therapy using MSCs from specific anatomical sources. Based on the proteomics data of OM-MSCs-Exo we have obtained and the PPI network analysis of FGFR1, future studies could screen and validate other key exosomal components that are co-sorted with or regulated by FGFR1 (e.g., other receptor tyrosine kinases or non-coding RNAs), which may exert synergistic or complementary effects with FGFR1, forming a more complex regulatory network. Future research should include an examination of PLCγ1 phosphorylation and the dynamics of its downstream second messengers. Beyond the classical expression of inflammatory factors, the functional state of microglia is better reflected in their phagocytic capacity, synaptic pruning activity, and real-time interactions with neurons. Follow-up research could deeply investigate whether OM-MSCs-Exo^oe−FGFR1 can enhance the ability of microglia to clear Aβ or improve their synaptic protective function in pathological environments. Combined with bioinformatics analysis, changes in signaling pathways can be directly linked to more specific neuroprotective outcomes.

Conclusion

Overall, our findings provide evidence supporting the potential of OM-MSCs-Exo in alleviating cognitive deficits and neuroinflammation in a mouse model of AD. The data are consistent with a plausible mechanism wherein exosome-associated FGFR1 engages with PLCγ1 in microglia, thereby promoting a shift toward the anti-inflammatory M2 phenotype. While this study delineates a novel functional axis and offers a mechanistic framework, further investigation is required to fully elucidate the detailed molecular cascades and in vivo delivery dynamics. Collectively, this work may contribute to the understanding of OM-MSCs-Exo as a therapeutic candidate and offers a foundation for developing strategies targeting microglial polarization in neurodegenerative diseases.

Supplementary Information

Below is the link to the electronic supplementary material.

Funding

This work was supported by the National Natural Science Foundation of China (No. 82460268), the Hainan Province Clinical Medical Research Center (No. LCYX202410, LCYX202309), and the Hainan Provincial Postdoctoral Science Foundation.

Data Availability

Research data are not shared.

Declarations

Conflict of interest

The authors declare no competing interests.