Neuroprotective effects of Berberine-loaded BMSCs-apoptotic extracellular vesicles on retinal ganglion cells: therapeutic potential for glaucomatous injury

Abstract

Glaucoma is a leading cause of irreversible blindness, primarily driven by the progressive degeneration of retinal ganglion cells (RGCs). In this study, we report the novel application of bone marrow mesenchymal stem cells (BMSCs)-derived apoptotic extracellular vesicles (ApoEVs) in a glaucomatous ischemia/reperfusion (IR) model. ApoEVs exhibited remarkable anti-inflammatory properties, were efficiently internalized by retinal neurons, promoted RGC survival, and preserved visual function. Transcriptomic analysis revealed that ApoEV treatment significantly downregulated Irgm1, an inflammation-related gene. Notably, this study also established a new drug delivery strategy by successfully loading Berberine (Ber)—a natural compound with well-documented anti-inflammatory and neuroprotective effects—onto ApoEVs. The combination further enhanced their protective effects on RGCs, with synergistic suppression of inflammation and improved neuronal viability. Mechanistically, this enhancement was mediated through the coordinated inhibition of the MAPK signaling pathway via Irgm1, which is identified here for the first time as a potential therapeutic target in glaucoma. Collectively, our findings highlight the dual function of Berberine-loaded ApoEVs as a potent cell-free therapeutic strategy that integrates targeted anti-inflammatory and neuroprotective effects, offering a promising new avenue for the treatment of glaucomatous neurodegeneration.

Graphical abstract

Supplementary Information

The online version contains supplementary material available at 10.1186/s12951-025-03714-2.

Introduction

Glaucoma remains the leading cause of irreversible blindness worldwide. According to the World Health Organization (WHO), approximately 76 million individuals were affected by glaucoma in 2020, with over 10% progressing to complete blindness. This number is projected to rise to 111.8 million by 2040 [1]. The resulting vision loss imposes considerable physical, psychological, and socioeconomic burdens on both patients and society. The hallmark pathological feature of glaucoma is the progressive and selective degeneration of retinal ganglion cells (RGCs) [2]. Elevated intraocular pressure (IOP) is the primary risk factor for glaucoma, and the resultant retinal ischemia/reperfusion (IR) injury is a key contributor to RGC death [3].

Mesenchymal stem cells (MSCs) are multipotent stromal cells capable of differentiating into various mesodermal lineages, including osteoblasts, chondrocytes, myocytes, and adipocytes [4]. Due to their potent immunomodulatory and regenerative capacities, MSCs have been extensively studied in the context of cell-based therapy [5, 6]. In recent years, MSC-derived secretory products—particularly extracellular vesicles (EVs)—have garnered attention as a promising cell-free therapeutic approach with low immunogenicity for treating inflammatory and degenerative diseases [7, 8]. EVs are heterogeneous membrane-bound nanoparticles released by nearly all cell types and are essential mediators of intercellular communication in both physiological and pathological settings [9]. Increasing evidence suggests that EVs, either as therapeutic agents themselves or as drug delivery vehicles, may represent a novel and effective strategy for the treatment of glaucoma [10–12].

A growing body of evidence indicates that programmed cell death pathways critically regulate RGC degeneration in glaucoma [13]. The proper regulation and clearance of dying cells are essential for maintaining immune homeostasis and preventing pathological inflammation or malignancy. Among these pathways, apoptosis plays a fundamental role in tissue homeostasis by eliminating superfluous, damaged, or immunogenic cells during development and immune responses [14]. Apoptosis, traditionally associated with cell death and tissue damage, is now understood as a tightly regulated physiological process that contributes to tissue equilibrium and can potentially elicit reparative or anti-inflammatory responses through the release of bioactive components [15, 16].During apoptosis, cells release a distinct subtype of extracellular vesicles known as apoptotic extracellular vesicles (ApoEVs), which carry nuclear, cytoplasmic, and membrane-derived components—including nucleic acids, proteins, and lipids. These vesicles can be internalized by nearby or distant recipient cells, thereby exerting functional regulatory effects [17, 18]. Among them, MSC-derived ApoEVs have garnered attention as potent mediators of tissue regeneration and repair [19, 20]. For example, MSC-ApoEVs have been shown to facilitate regeneration across various tissues, including skin, hair follicles, bone, muscle, and vasculature [21, 22]. Mechanistically, they enhance hair follicle growth by activating the Wnt/β-catenin signaling pathway in dermal papilla cells [20], and promote bone formation in osteoporotic models via the Ras/Raf1/Mek/Erk signaling cascade [23]. Moreover, MSC-ApoEVs exhibit therapeutic potential in modulating inflammation and immune responses [24, 25]. However, the role of MSC-ApoEVs in ocular disease—particularly in neuroprotection against glaucomatous injury—remains largely unexplored.

In this study, we hypothesize that bone marrow-derived MSCs (BMSCs) ApoEVs exert neuroprotective effects on RGCs in a glaucomatous IR injury model. We observed that ApoEVs distributed efficiently into the retina and were internalized by endogenous retinal cells, including neurons. Treatment with ApoEVs significantly enhanced RGCs survival and functional integrity, thereby attenuating glaucomatous neurodegeneration. Transcriptomic analysis of retinal tissues treated with ApoEVs identified a set of differentially expressed genes, among which Irgm1 emerged as a key target. Irgm1 has been implicated in modulating immune responses and maintaining cellular homeostasis in the central nervous system, and its down-regulation may contribute to the observed neuroprotection [26, 27].

To further enhance the therapeutic efficacy of ApoEVs, we explored the co-administration of Berberine, a natural isoquinoline alkaloid derived from Coptis chinensis Franch, known for its potent anti-inflammatory properties [28]. Previous studies have shown that Berberine can suppress IRGM, the human immunity-related GTPase family M gene [29]. In addition, it was reported that in the mouse atherosclerotic plaque model, Irgm1 (the murine homolog of human IRGM) deficiency suppresses macrophage apoptosis by inhibiting ROS generation and MAPK signaling transduction [26]. In our model, intravitreal Berberine administration alone promoted RGCs survival. More importantly, its combination with ApoEVs resulted in a synergistic protective effect, significantly enhancing RGCs survival following IR injury.

Taken together, these findings provide compelling evidence for the therapeutic potential of Berberine-loaded BMSCs-derived ApoEVs in neuroprotection against glaucomatous injury. This work lays a theoretical foundation for the future development of ApoEV-based interventions in glaucoma and other retinal neurodegenerative diseases.

Methods and materials

Cell lines and cell culture

Isolation and primary culture of human bone marrow-derived cells

Bone marrow aspirates were obtained from five patients undergoing spinal fracture fixation or hip arthroplasty at Xiangya Hospital, Central South University. All procedures were approved by the Institutional Ethics Committee of Xiangya Hospital (Approval number: 2024091259), and written informed consent was obtained from all participants. Aspirates were centrifuged at 110 g for 5 min at room temperature, and the supernatant was discarded. The cell pellets were treated with red blood cell lysis buffer (Cat.AWC0358a, Abiowell) and then resuspended in high-glucose DMEM(Cat. C11995500BT, Gibco). After 24 h, the floating cells were washed away, and the medium was changed with F12(Cat.C11330500BT, Gibco), which contained 15% FBS (Cat.FSP500, ExCell), and 1% penicillin-streptomycin (Cat. C100C5, Ncmbio) after 48 h.

R28 cell culture

R28 cells, a retinal precursor cell line with differentiation potential, are commonly employed in vitro to investigate neuroprotective mechanisms and the pathophysiology of RGCs. It was provided by the Department of Anatomy and Neurobiology of Central South University (Changsha, China). Cells were cultured in low-glucose DMEM (Cat.C11885500BT, Gibco) supplemented with 10% FBS (Cat. FSP500, ExCell) and 1% penicillin-streptomycin (Cat.C100C5, Ncmbio) at 37 °C in a humidified atmosphere containing 5% CO₂.

Induction of BMSCs apoptosis

Undifferentiated human BMSCs were washed twice with phosphate-buffered saline (PBS, Cat.G4202, Servicebio), after which the culture medium was replaced with complete medium containing 250 nM staurosporine (STS, Cat.HY-15141, MCE) and FBS depleted of exosome (Cat.C38010050.VivaCell). Cells were incubated for 12 h to induce apoptosis. Apoptosis was confirmed by morphological assessment and Western blot analysis.

Isolation and characterization of apoptotic extracellular vesicles (ApoEVs)

ApoEVs were isolated according to previously established protocols [30]. Briefly, 12 h after STS-induced apoptosis, the culture supernatant was collected and subjected to centrifugation at 800 × g for 10 min to remove cellular debris. The supernatant was then centrifuged at 16,000 × g for 30 min to pellet ApoEVs, which were subsequently washed twice with filtered PBS. Protein concentration was determined using a BCA protein assay kit (Cat.WB6501.Ncmbio) for quantification purpose.

Nanoparticle tracking analysis (NTA) was performed using a NanoSight NS300 instrument (Malvern Instruments) equipped with a 405 nm laser and an sCMOS camera to assess size distribution. ApoEV samples were resuspended and diluted in filtered PBS. Each sample was captured for 15 s using a camera level of 14 and a detection threshold of 8, and measurements were performed in triplicate. Filtered PBS alone was imaged to confirm the absence of contaminating particles. A total of 1,498 frames were captured and analyzed using NTA 3.2 Dev Build 3.2.16 software (Malvern Instruments).

To assess apoptotic markers, purified ApoEVs were analyzed by Western blotting using antibodies against cleaved caspase-3 and β-actin. Additionally, ApoEVs were stained with Annexin V-Fluos and examined under a confocal laser scanning microscope. Furthermore, the expression of phosphatidylserine (PS) on the vesicle surface was assessed by Annexin V labeling using a nanoflow cytometry system (Flow NanoAnalyzer, Xiamen Folo Biotechnology Co., Ltd., China). Briefly, purified ApoEVs were incubated with fluorophore-conjugated Annexin V in binding buffer according to the manufacturer’s instructions. After incubation, samples were analyzed directly without further washing. The nanoflow cytometer was calibrated using standardized 100 nm and 200 nm reference beads, and data acquisition was performed under optimized threshold and gain settings. Annexin V-positive events were quantified and expressed as a percentage of total vesicles. The enrichment of Annexin V on ApoEVs confirmed their apoptotic vesicle identity.

Scanning electron microscopy (SEM) and transmission electron microscope(TEM)

SEM

ApoEVs in PBS were fixed with 2.5% glutaraldehyde at 4 °C for 2 h, followed by post-fixation with 1% osmium tetroxide(Ted Pella Inc, Product Number.18456). Samples were dehydrated through graded ethanol and acetone series, then subjected to tert-butyl alcohol and isoamyl acetate replacement. After vacuum drying, vesicles were mounted on conductive tape, gold-coated, and examined using a Hitachi S-3400 N scanning electron microscope.

TEM

ApoEVs were fixed in 2.5% glutaraldehyde (0.1 M phosphate buffer, pH 7.4) at 4 °C for at least 2 h, followed by post-fixation in 1% osmium tetroxide for 1–2 h. After phosphate-buffered rinses, samples were dehydrated through a graded acetone series (50%, 70%, 90%, and 100%, each concentration for 15 min), then infiltrated with a mixture of acetone and embedding resin(1: 1), and embedded in pure resin for 12 h. Polymerization was performed at 37 °C overnight and then at 60 °C for 12–24 h. Ultrathin Sects. (50–100 nm) were cut using a Leica UC-7 ultramicrotome, stained with uranyl acetate and lead citrate, and examined using a Hitachi HT7700 transmission electron microscope.

Labeling and tracking of ApoEVs

The protein concentration of ApoEVs was first determined using a BCA protein assay kit. A fluorescent dye (Cat.40758ES25, Yeasen) was then added to the ApoEV suspension and incubated for 10 min at room temperature. After incubation, 10 mL of 1× PBS was added to the ApoEV–dye mixture to dilute unbound dye, followed by gentle mixing. The labeled ApoEVs were re-isolated using the same centrifugation protocol as previously described to remove excess dye. The final pellet was resuspended in 200 µL of 1× PBS to obtain the fluorescently labeled ApoEVs.

For in vitro tracking, R28 cells were incubated with the labeled ApoEVs for 24 h. The cells were then fixed and processed for immunofluorescence staining according to standard protocols. Cellular uptake of labeled ApoEVs was visualized under a fluorescence microscope.

For in vivo tracking, 1.5 µL of the labeled ApoEV suspension was intravitreally injected into the vitreous cavity. After 72 h, mice were sacrificed, and retinas were dissected and flat-mounted. The distribution of labeled ApoEVs was observed using a confocal laser scanning microscope.

Western-blot

Retinal tissues were harvested at the indicated time points, and total protein was extracted using RIPA lysis buffer (Cat.WB3100, Ncmbio) supplemented with a protease and phosphatase inhibitor cocktail (Cat.P002, Ncmbio) (100:1, v/v). Protein concentration was determined using a BCA protein assay kit. Equal amounts of protein were separated on 8%, 10%, or 15% SDS-PAGE gels, depending on molecular weight, and subsequently transferred onto polyvinylidene difluoride (PVDF) membranes.

Membranes were blocked with 5% skim milk or bovine serum albumin (BSA, Cat.4240, Biofroxx) in in PBS with 0.1%Triton X-100 (PBST, Cat.T8200, Solarbio) for 2 h at room temperature, followed by overnight incubation at 4 °C with primary antibodies. After five washes with PBST (5 min each), membranes were incubated with appropriate HRP-conjugated secondary antibodies (Cat.SA00001-1or SA00001-2,Proteintech)for 2 h at room temperature. Protein bands were visualized using enhanced chemiluminescence (ECL, Cat.P10100, Ncmbio) detection reagents and quantified using ImageJ software (National Institutes of Health, Bethesda, MD, USA).

The following antibodies were used in this study: p44/42 MAPK (Erk1/2) antibody (Cat. #4695, Cell Signaling Technology), phospho-p44/42 MAPK (Erk1/2) antibody (Cat. #9101, Cell Signaling Technology), phosphor-p38 antibody (Cat. #4511, Cell Signaling Technology), p38 antibody (Cat. #8690, Cell Signaling Technology), IRGM1(Cat. #14979, Cell Signaling Technology), and ACTIN (Cat. 66009-1, Proteintech).

qRT-PCR

Total RNA was isolated from retinal tissues using Trizol reagent, and RNA concentration/purity was assessed spectrophotometrically. High-quality RNA samples were reverse-transcribed into cDNA using a cDNA Synthesis SuperMix kit with 1 µg total RNA as template (Cat.11141, Yeasen). Target gene amplification was performed using SYBR Green Master Mix (Cat.11202, Yeasen) under manufacturer- recommended conditions, with β-ACTIN serving as the internal control. Primer sequences are provided in Supplementary Table 1.

Cell viability assay

The Cell Counting Kit-8 (Cat. EK-5103; ECOTOP SCIENTIFIC) was applied to measure the viability of cells according to the manufacturer’s instructions. R28 cells were seeded in 96-well plates at a density of 5000 cells/well and incubated for 24 h. Then, they were divided into four groups: Ctrl/PBS group, Ctrl/ApoEV group, H₂O₂ treatment group, and H₂O₂ + ApoEV treatment group. The H₂O₂ treatment group was cultured in the medium containing H₂O₂ (200 µmol, Cat. 10011218,Sinopharm Chemical Reagent Co.,Ltd) for 2–4 h. Subsequently, cell absorbance was measured at 450 nm using a microplate reader (Synergy LX; BioTek, USA) after 2–4 h of CCK8 addition.

Calcein AM and Propidium iodide (PI) Live/Dead cell staining

R28 cells were seeded into 24-well plates and cultured under standard conditions. After the designated treatments, the culture medium was removed, and cells were gently washed once with PBS. A fresh staining solution containing Calcein acetoxymethyl ester (Calcein AM) and propidium iodide (PI) (Cat.KGAF001, KeyGEN Biotech), each diluted 1:2000 in low-glucose basal medium, was prepared. Cells were incubated with the staining solution at 37 °C in the dark for 30 min. After incubation, the staining solution was removed and replaced with PBS. Fluorescence images were then captured using a fluorescence microscope (Leica DMi8, Wetzlar, Germany) to assess cell death in each group.

Intracellular reactive oxygen species (ROS) detection

The supernatant of each group of cells was discarded, and the cells were washed twice with serum-free medium. DCFH-DA (Cat.E-BC-K138-F, Elabsciences)was diluted with serum-free medium at a ratio of 1:1000 to a final concentration of 10µM and added to the cells. The cells were incubated in the dark at 37 °C in a cell culture incubator. After the treatment was completed, the cells were washed with serum-free medium 3 times. Fluorescence was detected using a fluorescence microscope (Leica DMi8, Wetzlar, Germany).

Animal model and drug administration

In this study, wild-type C57BL/6J mice (8 weeks old, weighing 20–25 g) were used as experimental subjects. Based on our previous research, an acute angle-closure glaucoma model was established by elevating IOP to 120 mmHg for 60 min [31, 32].

Mice were anesthetized via intraperitoneal injection of 1% pentobarbital sodium (10 mg/kg). Following anesthesia, 0.5% tropicamide phenylephrine (Santen Pharmaceutical Co, Ltd, Shiga Plant) followed by 0.4% obtained hydrochloride (Benoxil; Santen Pharmaceutical Co, Ltd) were then applied to the eyes for mydriasis and cornel topical anesthesia. The animals were placed on a heating pad to maintain body temperature at 37 °C. A 30-gauge needle connected to a saline infusion system was carefully inserted into the anterior chamber of the eye. The IOP was gradually increased to 120 mmHg and maintained for 60 min. In the control group, a sham procedure was performed without IOP elevation in the left eye.

Intravitreal drug delivery was conducted as previously described [31]. Using a Hamilton syringe equipped with a 30-gauge glass micropipette, intravitreal injections were performed under a surgical microscope. A total volume of 1.5 µL of PBS, ApoEVs, Berberine (Ber), or ApoEVs co-incubated with berberine (Ber-ApoEVs) was slowly injected into the vitreous cavity. The IR model was induced 24 h after injection.

All animal procedures were conducted in accordance with the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research. The experimental protocols were reviewed and approved by the Institutional Animal Care and Use Committee of the Laboratory Animal Research Center, Xiangya hospital, Central South University (Approval No:2024091259).

Histological staining and retinal morphometry

Histological evaluations were performed on retinal cross-sections. H&E-stained retinal morphology was acquired post IR injury, to visualize and obtain a count of cells in the ganglion cell layer (GCL). Eyes were immediately enucleated post-execution and fixed in FAS eye fixation fluid (Servicebio, China) for 24 h at 4 °C. The eyes were embedded in paraffin(Servicebio, Cat No. G1101) and were cut into sections of 4 μm thickness through the optic nerve, prepared in the standard way, then stained with hematoxylin and eosin [33]. Retinal morphology was observed, and photomicrographs were taken using a light microscope. Sections containing the optic nerve stump were chosen for consistency, and at least three discontinuous sections (per animal) were analyzed using CaseViewer software.

Whole-mount retina immunostaining

Mouse eyes were enucleated and fixed in 4% paraformaldehyde (PFA, w/v) for 2 h at room temperature. After removing the cornea and lens, the retinas were further fixed in 4% PFA for an additional 1 h at RT. The retinas were then dissected, flat-mounted onto glass slides, and divided into four equal quadrants. Following permeabilization with 0.3% PBST for 10 min, the tissues were blocked with 5% BSA for 1 h and incubated overnight at 4 °C with an anti-Brn3a antibody (Cat.ab245230, Abcam,1:500 dilution). After three PBS washes, the retinas were incubated with Alexa Fluor 488-conjugated goat anti-rabbit secondary antibody(Cat.#4412, Cell Signaling Technology) for 2 h at RT, followed by mounting.

Fluorescence microscopy was used to capture images of each retinal quadrant (with a shared central region). Brn3a-positive cells were counted using ImageJ to determine RGCs density (RGCs/mm²). The mean RGCs density across all groups was calculated, and relative RGCs density was expressed as a percentage of the control group. Statistical analysis of Brn3a + cell density was performed using GraphPad Prism 9.0.

VEP&ERG

Visual electrophysiological assessments

Flash visual evoked potential (FVEP)

Mice were anesthetized with 1% sodium pentobarbital, and then 0.5%tropicamide phenylephrine (Santen Pharmaceutical Co, Ltd, Shiga Plant) followed by 0.4%obtained hydrochloride (Benoxil; Santen Pharmaceutical Co, Ltd) were then applied to the eyes for mydriasis and cornel topical anesthesia.The animals were placed on a heating pad to maintain body temperature at 37 °C. GenTeal lubricant eye gel(GenTeal^® Tears Lubricant Eye Gel, Alcon, Fort Worth, TX, USA) were applied to prevent corneal dehydration. Three needle electrodes were subcutaneously inserted at the following locations:

Recording electrode

Midpoint between the ears, in contact with the occipital bone.

Reference electrode

Subcutaneous insertion at the nasal region.

Ground electrode

Subcutaneous insertion at the tail.

One eye was occluded, and FVEP recordings were performed following international standards (http://www.iscev.org/standards). The procedure was repeated for the contralateral eye. The N1-P1 amplitude was analyzed for each group.

Flash electroretinography (FERG)

Prior to testing, mice were dark-adapted for > 12 h. Preparation was similar to FVEP, except recordings were conducted under dim red light. Two corneal ring electrodes and three needle electrodes were used:

Recording electrodes

Placed on both corneas, with carboxymethylcellulose eye drops enhancing conductivity and moisture.

Reference electrodes

Subcutaneously inserted near the nasal region.

Ground electrode

Subcutaneously inserted at the tail.

Stimulation and recording followed the International Society for Clinical Electrophysiology of Vision (ISCEV) standards (http://www.iscev.org/standards). The amplitudes and implicit times of the a-wave and b-wave were analyzed.

mRNA-seq and bioinformatic analysis

1. Bulk RNA sequencing

The total RNA of mouse retina tissue was extracted using Trizol reagent (Invitrogen, Carlsbad, CA, USA). The concentration, quality, and integrity were determined using a NanoDrop spectrophotometer (Thermo Scientific). 3 micrograms of RNA were used as input material for the sequencing library preparation. Sequencing libraries were generated according to the following steps. The sequencing library was then sequenced on a NovaSeq 6000 platform (Illumina) by Shanghai Personal Biotechnology Cp. Ltd.

• b) Transcriptome analysis

The original data in FASTQ format (Raw Data) was filtered using fastp (0.22.0) software to get a high-quality sequence (Clean Data) for further analysis. The reference genome (Mus_musculus. GRCm39) and gene annotation files were downloaded from the genome website. The filtered reads were mapped to the reference genome using HISAT2 (v2.1.0). HTSeq (v0.9.1) statistics was used to compare the Read Count values on each gene as the original expression of the gene. RSEM (version 1.2.31) was used for the transcript abundance estimation. The following bioinformatic analysis is conducted in R (4.2.3). The RSEM-expected counts were then subjected to DESeq2 (1.38.3) [34] for the identification of differentially expressed genes (DEGs), with a criterion of |log2 fold change| >1 and adjusted p-value of < 0.05. Venn diagrams showing the number of common DEGs across the experimental comparisons were drawn by using Jvenn [35]. Meanwhile, ComplexHeatmap (v2.16.0) was used to perform a bidirectional clustering analysis of all different genes of samples [36]. A heatmap was generalized according to the expression level of the same gene in different samples and the expression patterns of different genes in the same sample with the Euclidean method to calculate the distance and the Complete Linkage method to cluster. ClusterProfiler (v4.6.0) software was used to carry out the enrichment analysis of the KEGG (Kyoto Encyclopedia of Genes and Genomes) pathway of differential genes, focusing on the significant enrichment pathway with a P-value < 0.05 [37]. Other R package used to declaratively creating graphics includes ggplot2 (3.4.4), cowplot (1.1.3), RColorBrewer (1.1.3), ggrepel (0.9.5) [38–40].

Preparation of ApoEV-Berberine complex (Ber-ApoEV)

ApoEVs were incubated with berberine (Ber) at a mass ratio of 1:1.5 (ApoEV protein: berberine) under gentle agitation at room temperature overnight. After incubation, unbound berberine was removed using the same isolation protocol as described above. The concentration of berberine encapsulated within ApoEVs was determined using high-performance liquid chromatography (HPLC) analysis conducted on an isocratic liquid chromatography system (JASCO), which included a dual pump (PU-1580), a Rheodyne injector with a 20 µL loop, and a photodiode array detector (MD-1510). Chromatographic data were recorded using Jasco-Borwin software version 1.50. Separation was performed on a Cosmosil C8 column (150 × 4.6 mm, 5 μm particle size) using a mobile phase consisting of water and acetonitrile (70:30, v/v) containing 0.2% trifluoroacetic acid, at a flow rate of 1.0 mL/min. The column was equilibrated prior to sample injection, and detection was carried out at a wavelength of 348 nm. The HPLC method was developed and validated according to ICH guidelines to ensure reliable quantification of Berberine.

Image processing using Imaris

Colocalization analysis and quantification of IBA-1 and OX-6 signals were performed using Imaris10.2.0 (Bitplane). Confocal z-stack images were first converted into Imaris file format, and Regions of Interest (ROIs) were defined based on IBA-1 + signals to identify microglial cells. A 3D surface rendering was generated for both IBA-1 and OX-6 using consistent parameters [41]. The “Coloc” module in Imaris was used to assess signal overlap within the ROIs, and the colocalized volume (IBA-1 + OX-6+) was quantified for each dataset. For microglial morphology analysis, sphericity of IBA-1 + cells was quantified by generating individual 3D surfaces, followed by automatic measurement of sphericity values using the “Statistics” function in Imaris 10.2.0. All output data were exported and subjected to statistical analysis using GraphPad Prism 9.0.

Statistical analysis

All data are presented as mean ± standard deviation (SD). Statistical analyses were conducted using GraphPad Prism 9.0 (GraphPad Software, San Diego, CA, USA). For comparisons between two groups, unpaired two-tailed Student’s t-test was used. For multiple group comparisons, one-way or two-way analysis of variance (ANOVA) followed by Tukey’s post hoc test was applied when data conformed to a normal distribution. Normality was assessed using the Shapiro–Wilk test. When data did not meet normality assumptions, nonparametric tests were applied. Specifically, the Kruskal–Wallis test followed by Dunn’s multiple comparison test was used for multiple non-normally distributed group comparisons. A P value of < 0.05 was considered statistically significant. All experiments were performed in triplicate or as otherwise indicated. Details of sample size and statistical tests used are provided in the figure legends.

Results

Isolation and validation of BMSCs-derived apoevs

To isolate and characterize ApoEVs derived from BMSCs(Fig. 1A), a combination of TEM, SEM, NTA, Western-blotting, confocal microscopy and flow cytometry was employed, following standard protocols previously described for EV analysis [23]. TEM & SEM imaging revealed that ApoEVs displayed the classical cup-shaped or spherical morphology (Fig. 1B and C). NTA demonstrated a peak-shaped curve, with most of the area under the curve in the characteristic ApoEV size range of 100–300 nm and a significant peak at around 200 nm (Fig. 1D).

Fig. 1.

Isolation, characterization, and uptake of BMSCs-derived ApoEVs (A) Schematic workflow illustrating the isolation and validation process of BMSCs-ApoEVs. (B) SEM image showing the spherical morphology of ApoEVs.(C) TEM image showing the characteristic cup-shaped structure of ApoEVs. (D) NTA revealing a bell-shaped size distribution with a peak around 200 nm, consistent with the typical size range of ApoEVs.(E) Western blot analysis showing cleaved caspase-3 expression in ApoEVs. (F) Flow cytometry analysis demonstrating Annexin V expression on the surface of ApoEV. (G) Immunofluorescence staining further confirming Annexin V positivity in ApoEVs. (H) In vitro uptake assay showing internalization of DiD-labeled ApoEVs by recipient cells. (I) In vivo retinal distribution of DiD-labeled ApoEVs after intravitreal injection, confirming localization in the neural retina. Scale bars as indicated

Furthermore, Western blotting revealed the presence of cleaved caspase-3,a hallmark of apoptosis in the ApoEV fraction but not in parental BMSCs, confirming the apoptotic origin of these vesicles (Fig. 1E). Flow cytometry analysis and immunofluorescence staining confirmed that BMSCs-ApoEVs express classical apoptotic marker Annexin V, a hallmark of membrane phosphatidylserine exposure during apoptosis (Fig. 1F and G). These molecular features collectively validate the vesicular identity and apoptotic nature of the isolated ApoEVs.

To evaluate the biodistribution and uptake potential of BMSCs-ApoEVs, vesicles were labeled with the lipophilic fluorescent dye DiD and subsequently applied to both in vitro cultures and in vivo via intravitreal injection. Fluorescence microscopy revealed robust DiD signal in recipient cells in vitro, as well as in retinal tissue following in vivo administration, confirming the efficient internalization and retinal penetration of BMSCs-ApoEVs (Fig. 1H and I). These findings support the notion that ApoEVs can effectively reach neural retinal cells and are bioavailable for mediating local functional effects.

Neuroprotective effects of apoevs in acute glaucoma

The neuroprotective potential of ApoEVs was first assessed in vitro using the R28 retinal precursor cell line subjected to the Hydrogen Peroxide (H₂O₂)-Induced Oxidative Stress injury model [42]. As outlined in the experimental workflow (Fig. 2A), R28 cells were treated with ApoEVs at various concentrations. Cell viability, evaluated via CCK-8 assay, demonstrated a significant, dose-dependent improvement in survival. The application of 1.5 µL of ApoEVs at these concentrations resulted in pronounced enhancement in cell viability (Fig. 2B). ROS levels measured after H₂O₂ stimulation were markedly decreased in ApoEV-treated cells relative to PBS controls, indicating that ApoEVs mitigate oxidative stress-induced injury (Fig. 2C and D). ApoEV treatment significantly reduced PI-positive cell proportions from 59.32 ± 17.10% to 8.56 ± 7.23%, representing an 85.6% decrease in cell death following H₂O₂ exposure. Consistent findings were observed in PI staining assays, with the ApoEV-treated groups exhibiting reduced cell death at both 24 and 48 h post H₂O₂ stimulation compared to the untreated controls: Compared to the H₂O₂-injured group (H₂O₂/PBS, mean ± SD: 59.32 ± 17.10%), ApoEV treatment significantly reduced PI-positive cell percentages (H₂O₂/ApoEV, mean ± SD: 8.56 ± 7.23%), reflecting an 85.57% decrease in cell death rate. (Figure 2E and F).

Fig. 2.

BMSCs-ApoEVs protect R28 retinal precursor cells from oxidative stress-induced injury in vitro. (A) Schematic illustration of the experimental workflow. (B) CCK-8 assay in cell viability following ApoEV treatment. (C, D) Quantification and representative images of intracellular reactive oxygen species (ROS) levels. (E, F) Propidium iodide (PI) staining assay results at 2 and 4 h post H₂O₂ exposure. Data are presented as mean ± SD from at least three independent experiments. Statistical analysis was performed following a normality test; for non-normally distributed data, the Kruskal–Wallis test followed by pairwise comparisons was used to determine significance. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. Scale bars as indicated

In vivo, the neuroprotective efficacy of ApoEVs was evaluated in a murine model of acute glaucoma induced by retinal IR injury. Intravitreal injections of ApoEVs were administered as depicted in the schematic diagram (Fig. 3A). To evaluate the neuroprotective effect of ApoEVs on retinal structure, HE staining was performed on retinal sections at 3 and 7days post-injury (dpi) (Fig. 3B-E). In the IR/PBS group, a marked reduction in total retinal cell number was observed compared to the Ctrl/PBS group, indicating significant retinal degeneration induced by IR injury. Notably, treatment with ApoEVs partially rescued this cell loss. Quantitative analysis revealed a significant increase in the number of retinal cells in the IR/ApoEV group compared to the IR/PBS group at both 3 dpi and 7 dpi (P < 0.05), suggesting that ApoEV administration mitigated IR-induced retinal damage and preserved cellular integrity. To further evaluate RGCs survival, retinal wholemounts were immune-stained for Brn3a at 3dpi and 7dpi. Quantitative analysis demonstrated a significantly higher RGCs survival rate in the ApoEV-treated group compared to IR group, as compared to the IR injury group at 3dpi (PBS/IR, mean ± SD: 60.65 ± 5.27%), ApoEV treatment significantly increased RGCs survival (ApoEV/IR, mean ± SD: 77.84 ± 7.37%), corresponding to a 28.3% improvement in relative RGC viability. The result is consistent at 7dpi as RGCs survival in the untreated IR group (PBS/IR, mean ± SD: 28.82 ± 6.11%) was significantly increased in the ApoEV-treated group (ApoEV/IR, mean ± SD: 63.46 ± 10.05%) (Fig. 3F-I). The above data indicated that ApoEVs confer robust neuroprotection against ischemic damage.

Fig. 3.

BMSCs-ApoEVs confer structural and functional neuroprotection in a murine retinal IR model (A) Schematic of intravitreal ApoEV administration in the IR model. (B, C) Representative images of H&E staining and quantification at 3 days post-reperfusion(3dpi). (D, E) Representative images of H&E staining and quantification at 7 days post-reperfusion(7dpi). (F, G) Brn3a immunostaining of retinal wholemounts at 3 dpi. (H, I) Brn3a immunostaining of retinal wholemounts at 7 dpi. (J–N) Functional assessments using ERG and VEP. Statistical analysis was performed using the Kruskal–Wallis test with pairwise comparisons after confirming data distribution. All data are presented as mean ± SD; *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. Scale bars as indicated

In addition to assessing the morphological changes, retinal visual function was evaluated using F-ERG and F-VEP. The combined application of these two techniques provides a more comprehensive assessment of retinal functional status. F-ERG, which reflects retinal function, showed that both a-wave and b-wave amplitudes were significantly decreased following IR injury, whereas treatment with intravitreal ApoEV administration led to a recovery of these amplitudes (Fig. 3J–L). Furthermore, F-VEP was employed to objectively evaluate the functionality of the retinal–cortical neural pathway. The IR group exhibited a significant reduction in N1–P1 amplitude, indicating impaired functional output of RGCs. Notably, ApoEV treatment partially restored the N1–P1 amplitude compared to the IR group, suggesting a protective effect of ApoEVs on RGCs function and their contribution to maintaining normal visual signal transmission (Fig. 3M and N).

Collectively, these data highlight the therapeutic potential of MSC-ApoEVs in preserving retinal structure, reducing apoptosis, and restoring neuronal function in acute glaucoma models.

Transcriptomic profiling reveals ApoEV-mediated regulation of neuroinflammatory responses in IR-injured retina

To investigate the molecular mechanisms underlying the neuroprotective effects of ApoEVs in retinal IR injury, we performed bulk RNA sequencing on retinal tissues collected from Ctrl/PBS, IR/PBS, and IR/ApoEV groups. Violin plots of transcript abundance (TPM) demonstrated consistent data quality across biological replicates (Fig. 4A). DEG analysis revealed substantial transcriptional changes induced by IR, with 1,630 upregulated and 631 downregulated genes in IR/PBS versus Ctrl/PBS (Fig. 4B). Notably, ApoEV treatment partially reversed this expression profile, with 458 genes upregulated and 67 downregulated in IR/ApoEV versus IR/PBS.

Fig. 4.

Transcriptomic analysis reveals that ApoEV treatment modulates IR-induced immune responses (A) Violin plot showing the distribution of gene expression (TPM) across replicates from each group, demonstrating data quality and consistency. (B) Bar graph summarizing the number of differentially expressed genes (DEGs) across comparisons. DEGs were defined as those with adjusted p-value < 0.05 and absolute log₂ fold change > 1. (C, D) Volcano plots illustrating significantly altered genes between groups. Notably, Irgm1 was highly upregulated in IR/PBS vs. Ctrl/PBS and downregulated following ApoEV treatment (IR/PBS vs. IR/ApoEV). (E, F) Heatmaps displaying top DEGs associated with inflammatory and apoptotic responses. Rows represent genes, columns represent biological replicates. Color scale indicates Z-score normalized expression (red: high expression, blue: low expression) (G, H) GO enrichment analysis of DEGs in IR/PBS vs. Ctrl/PBS (G) and IR/PBS vs. IR/ApoEV (H). (I, J) KEGG pathway analysis revealing upregulation of MAPK signaling pathway and apoptosis in IR/PBS group, which were partially reversed by ApoEV administration

Volcano plots further highlighted these differences, showing robust upregulation of immune and apoptosis-related genes such as Irgm1, Tnfaip3, and Cxcl10 in the IR/PBS group [43], which were significantly downregulated following ApoEV intervention (Fig. 4C and D). Heatmap clustering confirmed the normalization of these inflammatory gene signatures in the IR/ApoEV group, including marked suppression of Irgm1, a key regulator of inflammation and autophagy under stress conditions (Fig. 4E and F).

Gene Ontology (GO) enrichment analysis revealed that IR insult activated numerous immune-related biological processes, including “immune system process,” and “response to cytokine stimulus” (Fig. 4G, Sup Fig. 1A-1D). These responses were largely attenuated in the IR/ApoEVs group, which exhibited reduced enrichment of innate immune terms and increased expression of genes related to cellular homeostasis (Fig. 4H).

Similarly, KEGG pathway analysis showed that IR triggered pathways related to inflammation and apoptosis, especially MAPK signaling (Fig. 4I, Sup Fig. 1E&1 F). Following ApoEVs treatment, activation of these pathways was significantly dampened (Fig. 4J). In particular, the MAPK signaling cascade, known to mediate stress and apoptotic responses in retinal neurons, was significantly downregulated, supporting the anti-inflammatory and neuroprotective effects of ApoEVs.

Collectively, these transcriptomic findings suggest that ApoEVs mitigate IR-induced retinal injury by suppressing immune and apoptotic signaling, particularly via the inhibition of Irgm1 expression and MAPK pathway activation.

ApoEVs impresses neuroinflammation in acute glaucoma

To validate the transcriptomic alterations observed in our RNA-seq analysis, we first examined the expression of Irgm1, a key regulator of immune responses and autophagy. Both Western Blot (Fig. 5A and B) and qRT-PCR(Fig. 5C) analyses demonstrated a marked upregulation of IRGM1/Irgm1 in the retina following IR injury.

Fig. 5.

BMSCs-derived ApoEVs mitigate neuroinflammation and microglial activation in the IR-injured retina. (A,B) Western blot analysis and quantification of IRGM1 protein expression in retinas after IR injury.(C) qRT-PCR analysis of Irgm1 mRNA levels after IR injury at several timepoints. (D) qRT-PCR analysis of Il-6, Caspase1, Igf1, Bcl2, and Bst2 mRNA levels in retinal tissues at 3 dpi. (E,F) Representative immunofluorescence images showing IBA-1 and OX-6 staining in the retina. Scale bar = 20 μm.(G) Quantification of percentage of OX-6stained ROI co-localized with IBA-1⁺ microglial cell in each group. (H,I) 3D morphological reconstruction and analysis of IBA-1⁺ microglial sphericity using Imaris software (v10.2.0). All quantitative data are shown as mean ± SD. Group comparisons were analyzed using the Kruskal–Wallis test followed by Dunn’s multiple comparisons. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. Scale bars as indicated

Building upon this observation, we sought to assess whether ApoEVs impact the broader neuroinflammatory environment in the retina. We conducted qRT-PCR analysis of retinal tissue harvested 3dpi to quantify expression of selected inflammation-related cytokines. Among these, Il-6, Igf1, Caspase1, and Bcl2 [44–47] were chosen for their established roles in regulating immune responses, apoptosis, and cellular stress in neural tissue(Fig. 5D). ApoEVs treatment resulted in a significant downregulation of Il-6 and Caspase1, both of which are typically associated with pro-inflammatory and pro-apoptotic signaling. In contrast, the levels of Igf1 and Bcl2 were significantly upregulated, consistent with enhanced neuroprotective and anti-apoptotic signaling. The expression of Bst2, a multifunctional cytokine-like molecule implicated in innate immune regulation, also exhibited a trend toward normalization following ApoEV treatment (Fig. 5D).

To further examine the effect of ApoEVs on microglial activation, we performed immunofluorescence staining using IBA-1 and OX-6 to label microglial cells in vivo (Fig. 5E and F). Quantitative analysis revealed a significant reduction in the number of IBA-1⁺ microglia in the retina of ApoEV-treated animals compared to vehicle-treated controls (Fig. 5G) indicating reduced microglial recruitment or proliferation in response to injury. In addition, using Imaris 10.2.0 software, we performed detailed 3D morphometric analysis to quantify the morphology change of IBA-1⁺ microglia(Fig. 5H and I). These analyses showed that IR injury markedly altered microglial morphology toward a more amoeboid, activated state, characterized by increased sphericity and reduced branching. ApoEVs treatment significantly reversed these changes, as reflected by a reduction in sphericity and restoration of a more ramified morphology. These findings suggest that microglial activation and morphological changes are key components of the IR-induced neuroinflammatory response, which can be attenuated by ApoEV treatment.

Collectively, these findings demonstrate that ApoEVs mitigate neuroinflammation after IR injury by downregulating pro-inflammatory cytokines, enhancing neuroprotective gene expression, and reducing microglial activation.

Protective effects of Berberine on RGCs survival following retinal IR

Berberine has been implicated in modulating inflammatory responses via IRGM1, raising the possibility of its relevance to neurodegenerative diseases such as glaucoma. To further investigate the neuroprotective effects of Berberine in the context of acute glaucoma, we administered Berberine via intravitreal injection in a mouse model and assessed its therapeutic impact (Fig. 6A).

Fig. 6.

Protective effects of Berberine on RGCs survival following retinal IR injury and characterization of Berberine-loaded ApoEVs (Ber-ApoEVs). (A) Experimental schematic depicting intravitreal injection of Berberine. (B) Representative H&E stained retinal sections after Berberine treatment at different doses. (C) Quantification of percentage of GCL cell number compared to Ctrl/PBS group from H&E stained sections. (D) Representative immunofluorescence images of Brn3a-positive RGCs in whole mounts after Berberine treatment, scale bar = 100 μm. (E) Quantitative analysis of Brn3a + RGCs density compared to Ctrl/PBS group. (F) Molecular docking model predicting stable binding of Berberine within the active site of IRGM1. (G) Schematic workflow for loading Berberine into ApoEVs. (H) HPLC quantification of Berberine concentration. (I) NTA showing particle size distribution and concentration of Ber-ApoEVs. (J) TEM images of Ber-ApoEVs. All quantitative data are shown as mean ± SD. Group comparisons were analyzed using the Kruskal–Wallis test followed by Dunn’s multiple comparisons. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001

A dose-response evaluation was first performed to determine the optimal Berberine concentration. H&E staining revealed that Berberine treatment preserved cell density of the inner retinal layers, particularly the GCL, which is typically reduced in following IR injury (Fig. 6B and C). As shown in Fig. 6D and E, Brn3a immunostaining revealed a significant preservation of RGCs following treatment with Berberine at a dose of 2.5 mM, compared to controls.

We further performed molecular docking analysis to predict the potential interaction between Berberine and IRGM1, which revealed that Berberine stably fits into the active pocket of IRGM1, forming multiple hydrogen bonds and hydrophobic interactions, suggesting a potential direct regulatory effect on IRGM1 function (Fig. 6F).

Taken together, these results demonstrate that intravitreal administration of Berberine provides significant structural and functional protection to RGCs in the IR-induced acute glaucoma model, likely through modulation of IRGM1-related pathways and inhibition of apoptosis.

Neuroprotection of apoevs combined with Berberine (Ber-ApoEVs) contributes to treatment of IR injury

Given the individual neuroprotective properties of ApoEVs and Berberine, we hypothesized that their combination might exert additive effects in attenuating retinal IR injury. Based on the shared modulation of IRGM1—a central mediator of inflammation—we generated Ber-ApoEVs for intravitreal delivery.

Berberine was loaded into ApoEVs using a mild sonication protocol followed by ultrafiltration (Fig. 6G). Drug loading efficiency was determined via HPLC, revealing a final Berberine concentration in the Ber-ApoEV suspension (Fig. 6H). Successful encapsulation was confirmed by NTA, which showed no significant changes in size or particle concentration compared to native ApoEVs (Fig. 6I). TEM analysis confirmed the typical cup-shaped morphology of Ber-ApoEVs, comparable to native ApoEVs, with no signs of aggregation or structural damage (Fig. 6J).

Mice received intravitreal co-administration of Berberine and ApoEVs (as separate or combined Ber-ApoEVs) (Fig. 7A). H&E staining demonstrated superior preservation of retinal architecture, particularly in the GCL, in the combination group relative to single-agent groups. Cell density in GCL was significantly greater, suggesting effective structural protection against IR-induced damage (Fig. 7B and D). Furthermore, Brn3a immunostaining revealed that the combination treatment led to a significantly higher density of surviving RGCs compared to either ApoEVs or Berberine alone (Fig. 7C and E).

Fig. 7.

Combined neuroprotective effects of BMSCs-derived ApoEVs and Berberine in retinal IR injury. (A) Experimental timeline of intravitreal injection of ApoEVs, Berberine, or Ber-ApoEVs. (B) Representative H&E stained retinal sections. (C) Representative immunofluorescence images of Brn3a-positive retinal ganglion cells in treated groups. Scale bar = 100 μm. (D) Quantification of percentage of GCL cell number compared to Ctrl/PBS group from H&E stained sections at 3dpi. (E) Quantification of Brn3a-positive RGCs density of treatment groups compared to Ctrl/PBS group at 3dpi. (F) Representative VEP waveforms recorded at 3dpi. (G) Quantification of VEP signal amplitudes. (H,I) Western blot analysis and quantification of ERK1/2 and p-38 protein expression in retinas after treatments. (J) qRT-PCR analysis of inflammatory and neuroprotective gene expression levels in retina tissue across treatment groups. All quantitative data are shown as mean ± SD. Group comparisons were analyzed using the Kruskal–Wallis test followed by Dunn’s multiple comparisons. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001

Electrophysiological assessments using VEP further confirmed functional improvements. The combined treatment group exhibited the highest VEP signal amplitudes at 3dpi, suggesting improved retinal signal transmission and RGCs excitability (Fig. 7F and G).

At the molecular level, Western blot analysis showed inhibition of p38 after both ApoEV/Ber alone as well as combined treatment (Fig. 7H and I), while qRT-PCR indicated a more pronounced downregulation of Caspase1, Bst2 and Il-6, and a concomitant upregulation of Bcl-2 and Igf-1 in the combination group, compared to either treatment alone (Fig. 7J). These data suggest that Berberine and ApoEVs may act on overlapping but complementary anti-inflammatory and anti-apoptotic pathways to mediate retinal protection, particularly through the p38/MAPK pathway.

Collectively, these results demonstrate that Ber-ApoEVs offers enhanced neuroprotection in the IR-injured retina through structural preservation, functional rescue, and transcriptional modulation of key inflammatory and apoptotic regulators.

Discussion

Characteristics of apoevs compared to other extracellular vesicles

Apoptosis is a major form of programmed cell death, orchestrated by highly conserved molecular mechanisms that play a pivotal role in development, immune regulation, and tissue homeostasis, as well as in various pathological processes [14]. Notably, apoptosis has been consistently observed as a predominant mode of RGCs death in glaucoma, highlighting its relevance in disease progression [13]. During apoptosis, cells undergo a sequence of regulated events including membrane blebbing, caspase activation, chromatin condensation, and DNA fragmentation, ultimately leading to the formation of ApoEVs [48]. These vesicles carry fragmented DNA, RNAs, lipids, and specific proteins, and are actively released into the extracellular space, where they influence the behavior and fate of neighboring cells [49].

Human BMSCs represent a reliable and clinically relevant source of EVs, owing to their low immunogenicity, strong immunomodulatory capacity, and established regenerative potential, making them highly suitable for allogeneic therapeutic applications [50]. EVs can be broadly classified into exosomes, microvesicles, and ApoEVs, based on their size and biogenesis [51]. Among these, ApoEVs are the largest (ApoEVs: 50–5000 nm, exosomes: 50–150 nm, micro-vesicles: 50–1000 nm) [49]. Beyond size, they differ in origin, morphology, membrane composition, and cargo content [22]. While exosomes and micro-vesicles are typically released by viable cells through endosomal pathways or membrane budding, ApoEVs are uniquely released during apoptosis, often containing apoptotic markers such as phosphatidylserine [52]. Importantly, due to their derivation from dying cells, ApoEVs possess inherently lower immunogenicity compared to EVs from viable cells, reducing the risk of triggering detrimental immune responses when used therapeutically.

Interestingly, despite their origin from apoptotic processes, ApoEVs do not simply function as passive by-products of cell death; rather, they are active mediators of intercellular communication and tissue remodeling [22, 53]. This phenomenon may be attributed to the fact that apoptosis is a tightly regulated and non-inflammatory process, during which ApoEVs selectively package and transmit bioactive signals that facilitate immune resolution, cellular adaptation, and tissue repair. Emerging evidence has revealed their pleiotropic effects, including anti-tumor, anti-inflammatory, and tissue repair-promoting functions [20, 48, 54]. These seemingly paradoxical properties likely stem from their unique molecular cargo, which allows ApoEVs to convey regulatory signals that promote immune resolution and cellular regeneration, rather than propagating apoptosis. In this context, we investigated BMSCs-derived ApoEVs in a glaucoma-related IR model, focusing on their capacity to preserve RGCs viability.

Given previous studies highlighting the neuroprotective properties of EVs [55, 56], including ApoEVs, and considering that glaucoma is characterized by progressive neurodegeneration marked by the loss of RGCs, we investigated the potential of ApoEVs to preserve RGCs viability in a glaucoma-related IR model.

Regulation of inflammation by apoevs and the mechanism of action

Pathogenic mechanisms in glaucoma involve ischemia, oxidative stress, hypoxia, excitotoxicity, mitochondrial dysfunction and neuroinflammation [57, 58]. Based on what was recently been documented, modulating local immune responses and targeting inflammatory pathways could offer new therapeutic strategies for glaucoma management.

ApoEVs have been shown to enhance the expression of anti-apoptotic and pro-survival factors while simultaneously suppressing the activation of pro-inflammatory cytokines, which may play a critical role in mitigating the detrimental effects of IR injury in the retina. Our study found that ApoEVs promoted RGCs survival by modulating inflammatory responses, partly via targeting IRGM1, a protein intricately linked with both autophagy and immune regulation [59]. In disease models, IRGM1 exhibits context-dependent effects: while it negatively regulates NLRP3 inflammasome activation and maintains intestinal or immune homeostasis in certain inflammatory settings [60], its overexpression has also been associated with tissue damage, including enhanced oxidative stress and promoting MAPK activation [26, 27]. In our study, we found that IRGM1 expression was markedly upregulated following IR injury and exerted detrimental effects on the RGCs, suggesting it may serve as a pathological target mediating inflammation and cell death in glaucomatous neurodegeneration. While the role of IRGM1 has been extensively studied in various contexts, its function in glaucoma has not been previously reported. To our knowledge, this study provides preliminary evidence suggesting a potential role of IRGM1 in the pathophysiology of glaucoma.

In our study, ApoEVs could significantly modulate inflammatory pathways to promote RGCs survival. Specifically, pathway enrichment analysis based on our retinal RNA sequencing data suggests that ApoEVs may exert their effects, at least in part, by modulating the MAPK signaling pathway, which is critically involved in regulating inflammation, cell survival, and apoptosis [61, 62]. The MAPK pathway has been implicated in the progression of glaucomatous optic neuropathy, where it regulates the inflammatory response and cellular damage in RGCs [63, 64]. Moreover, A recent study demonstrated that MSCs- ApoEVs promote cartilage regeneration by modulating the immune microenvironment through MAPK signaling, supporting the notion that ApoEVs may exert anti-inflammatory effects via MAPK-related pathways [65]. The precise mechanisms through which ApoEVs regulate these pathways remain to be fully elucidated, but our results indicate that they exert protective effects by suppressing inflammatory damage and promoting cellular homeostasis.

Previous studies on ApoEVs have predominantly focused on their role in tissue regeneration and restoring homeostasis [17, 27]. However, our research highlights the anti-inflammatory effects of ApoEVs, suggesting their potential in mitigating neuroinflammation in other neurodegenerative diseases, such as Alzheimer’s disease and Parkinson’s disease. Future investigations should expand on these findings to explore the broader therapeutic applications of ApoEVs in modulating neuroinflammatory responses across various neurodegenerative disorders.

Synergistic effect of apoevs and Berberine in enhancing neuroprotection

In this study, the combination of ApoEVs with Berberine, a natural alkaloid with well-established neuroprotective properties, was found to enhance the protective effects against RGCs damage in the IR model. Berberine has been shown to exert neuroprotection by modulating inflammation, oxidative stress, and apoptosis [66, 67]. In addition to its neurological benefits, Berberine has emerged as a promising therapeutic agent for cardiovascular and metabolic diseases (CVMD), owing to its multi-target pharmacological activities validated in both basic and clinical studies [68]. Its anti-inflammatory mechanisms primarily involve the suppression of inflammatory cytokine production (e.g., IL-1β, IL-6, TNF-α) and inhibition of key signaling pathways such as NF-κB and MAPK signaling [28]. These findings provide important molecular insights into Berberine’s therapeutic potential in inflammatory diseases. In ophthalmologic research, Berberine has been reported to suppress inflammation and apoptosis via the MAPK pathway in dry eye disease [69], however, its role in glaucoma has not yet been reported.

When Berberine was co-incubated with ApoEVs, we observed a synergistic effect that was more pronounced than either treatment alone. This suggests that the two agents may act through complementary mechanisms. ApoEVs, by modulating inflammatory pathways and promoting cell survival, appear to work in concert with Berberine’s antioxidant and anti-inflammatory effects to enhance the overall neuroprotective response. The combination treatment may offer a promising therapeutic approach for conditions where neuroinflammation and cell death play a critical role, such as glaucoma.

This finding opens up the potential for further studies into the use of ApoEVs in combination with other pharmacological agents to maximize their therapeutic benefits. Exploring the mechanisms underlying this synergistic effect will be crucial for optimizing the clinical application of such combination therapies.

Translational potential of apoevs in clinical practice

The translational potential of ApoEVs in clinical settings is an exciting prospect for treating retinal diseases, particularly glaucoma. Given their ability to cross biological barriers and their immunomodulatory properties, ApoEVs could be used as a therapeutic strategy to promote cell survival and reduce inflammation in the retina. Current treatments for glaucoma, such as IOP-lowering drugs, do not directly target the underlying neurodegenerative processes or inflammation [70]. Thus, the development of therapies based on ApoEVs could provide a novel approach to preserving RGCs function and halting disease progression.

In addition to their use in glaucoma, ApoEVs may also have applications in other retinal diseases, such as diabetic retinopathy and age-related macular degeneration, where inflammation and retinal cell death are prominent features. The ability to deliver therapeutic molecules in a highly targeted manner using ApoEVs could also enhance the efficacy of treatment and minimize off-target effects.

However, several challenges remain in the clinical translation of ApoEVs. These include the efficient and scalable isolation of ApoEVs, as well as ensuring their stability, safety, and optimal delivery to target tissues [71, 72]. Furthermore, the potential for immune responses to ApoEVs must be carefully evaluated to ensure their safety in human patients.

Conclusion

In conclusion, our study demonstrates that BMSCs-derived ApoEVs exert significant neuroprotective effects in a glaucoma-related IR model, likely by modulating inflammatory responses and promoting RGCs survival. Mechanistically, these effects may involve the IRGM1/p-38/MAPK signaling axis, as suggested by our transcriptomic and validation analyses. Moreover, the synergistic benefits observed with the addition of Berberine indicate that combination therapies could further enhance the therapeutic potential of ApoEVs.

Future research should focus on further elucidating the molecular mechanisms underlying the protective effects of ApoEVs, particularly their interaction with inflammatory pathways. Additionally, exploring the clinical application of ApoEVs, both alone and in combination with other therapeutic agents, holds great promise for treating glaucoma and other neurodegenerative diseases. As the field of EVs continues to evolve, it is important to address the challenges related to their production, stability, and delivery to realize their full clinical potential.

Author contributions

M.Y: Writing – original draft, Validation, Methodology, Investigation, Formal analysis, Data curation, Conceptualization.X.Z Validation, Methodology, Investigation, Formal analysis. Z.Z: Methodology, Formal analysis.SQ.X: Validation, Methodology. J.C: Data curation. SJ.X: Visualization. X. M: Visualization.S.C: Writing – original draft, Validation, Formal analysis, Funding acquisition, Conceptualization. X.X: Writing – review & editing, Funding acquisition, Conceptualization, Supervision.

Funding

This work was supported by the National Key Research and Development Program of China(2024YFA1108700 and 2024YFA1108704), National Natural Science Foundation of China (Grant No. 82171058 to Xiaobo Xia, Grant No.82301201 to Si Chen, and Grant No. 82302711 to Shiqi Xiang), Postdoctoral Research Foundation of China (Grant No.2023M733945) to Si Chen, Natural Science Foundation of Hunan Province(Grant No.2025JJ60574to Si Chen, Grant No. 2023JJ40864 to Shiqi Xiang) the Youth Natural Science Foundation of Xiangya Hospital (No.2022Q05) to Si Chen, and the National Clinical Key Specialty of Ophthalmology.

Data availability

No datasets were generated or analysed during the current study.

Declarations

Ethics approval and consent to participate

All animals were treated according to the Association for Research in Vision and Ophthalmology Resolution on the Use of Animals in Research. All animal experiments were reviewed and approved by the Animal Care and Use Committee of the Laboratory Animal Research Center at the Xiangya Hospital of Central South University. All procedures were approved by the Ethics Committee of Xiangya Hospital, Central South University (Approval No. 2024091259). The informed consent was obtained for experimentation with human subjects. All authors listed consented with all relevant ethical regulations.

Competing interests

The authors declare no competing interests.