Exosomal miR-203 from bone marrow stem cells targets the SOCS3/NF-κB pathway to regulate neuroinflammation in temporal lobe epilepsy

Abstract

BACKGROUND

Epilepsy is a prevalent chronic neurological disorder affecting 50 million individuals globally, with temporal lobe epilepsy (TLE) being the most common form. Despite advances in antiepileptic drug development, over 30% of patients suffer from drug-resistant epilepsy, which can lead to severe cognitive impairments and adverse psychosocial outcomes.

AIM

To explore the role of bone marrow mesenchymal stem cell (BMSC)-derived exosomal miR-203 in the regulation of neuroinflammation in a mouse model of epilepsy, providing a theoretical basis for the development of targeted microRNA delivery therapies for drug-resistant epilepsy.

METHODS

Adult male C57BL/6 mice were divided into a control group and a TLE model of 30 mice each, and the TLE model group was established by injecting kainic acid. BMSCs were isolated from the mice, and exosomes were purified using ultracentrifugation. Exosomal miR-203 was identified and characterized using high-throughput sequencing and quantitative reverse-transcription polymerase chain reaction. The uptake of exosomes by hippocampal neurons and the subsequent effects on neuroinflammatory markers were assessed using in vitro cell culture models.

RESULTS

Exosomal miR-203 exhibited a significant upregulation in BMSCs derived from epileptic mice. In vitro investigations demonstrated the efficient internalization of these exosomes by hippocampal neurons, resulting in downregulation of suppressor of cytokine signaling 3 expression and activation of the nuclear factor kappaB pathway, ultimately leading to enhanced secretion of pro-inflammatory cytokines.

CONCLUSION

Our study identifies exosomal miR-203 as a key regulator of neuroinflammation in a mouse model of epilepsy. The findings suggest that targeting miR-203 may offer a novel therapeutic strategy for epilepsy by modulating the suppression of cytokine signaling 3/nuclear factor kappaB pathway, thus providing a potential avenue for the development of cell-free therapeutics.

Key Words: Epilepsy; Neuroinflammation; MiR-203; Exosomes; Bone marrow mesenchymal stem cells; Suppressor of cytokine signaling; Nuclear factor kappaB

Core Tip: This study highlights the role of bone marrow mesenchymal stem cell-derived exosomal miR-203 in regulating neuroinflammation in a mouse model of temporal lobe epilepsy. These findings suggest that miR-203 modulates the suppressor of cytokine signaling 3/nuclear factor kappaB pathway, leading to increased neuroinflammation. This pathway activation promotes the secretion of proinflammatory cytokines, contributing to the pathology of epilepsy. Targeting miR-203 may offer a novel therapeutic approach for drug-resistant epilepsy by reducing neuroinflammation and improving patient outcomes, offering potential for future cell-free therapeutic strategies.

INTRODUCTION

Epilepsy, a prevalent neurological disorder characterized by recurrent seizures, affects approximately 50 million people worldwide[1]. Temporal lobe epilepsy (TLE), the most common form of epilepsy, is associated with hyperexcitability of neurons, hippocampal damage, neuronal apoptosis, activation of astrocytes and microglia, neuroinflammation, oxidative stress, and cognitive impairment[2-4]. Despite advancements in antiepileptic drugs, over 30% of patients suffer from drug-resistant epilepsy (DRE)[5], which can lead to more severe memory and cognitive deficits as well as adverse psychosocial outcomes[6].

Neuroinflammation plays a critical role in the pathophysiology of epilepsy. Sustained inflammation exacerbates TLE through synaptic reorganization, mossy fiber sprouting, and neuronal damage, leading to recurrent seizures. The suppression of cytokine signaling 3 (SOCS3) has been implicated in the modulation of neuroinflammatory responses. SOCS3 is a negative feedback regulator of cytokine signaling that can inhibit the activation of the Janus kinase/signal transducer and activator of the transcription pathway, which is involved in the production of proinflammatory cytokines[7]. In the context of TLE, overactivation of neuroinflammatory pathways, such as the nuclear factor kappaB (NF-κB) pathway, can lead to a chronic inflammatory state that contributes to seizure progression and resistance to antiepileptic drugs[8]. While numerous basic and clinical studies have contributed to the development of antiepileptic drugs, a deeper understanding of the pathomechanisms by which TLE suppresses neuroinflammatory responses and apoptotic processes is needed, thereby improving patient memory and cognitive abilities and enhancing their quality of life.

MicroRNAs (miRNAs) play crucial roles in the differentiation, proliferation, and death of neuronal cells, and are known to regulate neurotransmitter release and neuroinflammatory responses[9-13]. In the context of epilepsy pathogenesis, miRNAs are thought to exert regulatory effects through the precise control of gene expression, influencing the behavior and interactions of neural cells[14-16]. Among various miRNAs implicated in neurological disorders, miR-203 has garnered attention because of its potential role in modulating neuroinflammation. The miR-203 targets SOCS3, a key negative regulator of cytokine signaling pathways[17]. SOCS3 inhibits the activation of the Janus kinase/signal transducer and activator of the transcription pathway, which is crucial for the production of proinflammatory cytokines such as tumor necrosis factor (TNF)-α and interleukin (IL)-6[18,19]. By binding to the 3’ untranslated region of SOCS3 mRNA, miR-203 can lead to the degradation of SOCS3 mRNA or block its translation, thereby reducing SOCS3 protein levels[20-22]. Downregulation of SOCS3 enhances the activation of proinflammatory pathways, contributing to the neuroinflammatory state associated with epilepsy. Understanding the interplay between miR-203 and SOCS3 could provide valuable insights into the molecular mechanisms underlying epilepsy and identify potential therapeutic targets for the management of neuroinflammation in epilepsy.

Exosomes are extracellular vesicles of endocytic origin that are important mediators of intercellular communication in the nervous system[23]. These nanoscale vesicles, measuring 30-100 nm in diameter, exhibit favorable characteristics, such as the ability to evade clearance by the reticuloendothelial system[24], cross the blood-brain barrier[25], and target specific cells within the central nervous system[26]. They have been implicated in the modulation of neuroinflammation and immune responses, and have potential as therapeutic agents against various neurological disorders[27].

This study investigated the role of exosomal miR-203 derived from bone marrow mesenchymal stem cells (BMSCs) in the regulation of neuroinflammation in a mouse model of epilepsy. We hypothesized that miR-203, which is differentially expressed in BMSC-derived exosomes from epileptic mice, modulates neuroinflammation by targeting the SOCS3/NF-κB pathway, a key regulator of the inflammatory response. By elucidating the mechanisms by which exosomal miR-203 influences neuroinflammation, this study provides a foundation for the development of targeted miRNA delivery strategies to improve outcomes in patients with DRE.

MATERIALS AND METHODS

Animal model and ethical approval

To establish a reliable animal model of epilepsy for studying the effects of exosomal miR-203 on neuroinflammation, 60 adult male C57BL/6 mice, aged 8-10 weeks and weighing 20-28 g, were randomly divided into two groups: A TLE model group and a control group, each consisting of 30 mice. The randomization process was conducted using a random number table to ensure that each mouse had an equal chance of being allocated to either group. Specifically, we assigned a unique number to each mouse and referred to a random number table to determine the group assignment. Mice with numbers corresponding to a randomly selected sequence were placed in the TLE model group, whereas the others were assigned to the control group. The TLE model group was injected with kainic acid at dose of 15-20 mg/kg to induce TLE. The Racine scale was used to quantify the severity of the seizures, allowing for a standardized assessment of epilepsy induction. All experimental procedures involving animals were conducted in strict accordance with the guidelines of the Institutional Animal Care and Use Committee of Dalian Medical University (No. JL-WW-2022120601) to ensure the ethical and humane treatment of the animals.

Racine scale

Epileptic seizures were classified according to Racine grading criteria, with grade 0 indicating the absence of convulsive seizures. Grade I indicates ear and face convulsions, whereas grade II indicates myoclonus without an upright position. Grade III denotes myoclonus in the orthostatic position. Grade IV represents generalized tetanic seizures and grade V represents tonic-clonic seizures accompanied by loss of postural control. In the epileptic status model, mice exhibiting class V or higher seizures lasting for 40 minutes were considered successful if they remained in good condition after resolution.

Isolation of BMSCs and exosome extraction

Bone marrow was extracted from the femurs of mice to ensure sterile conditions to maintain cell viability and purity. Extracted marrow was cultured in Dulbecco’s modified Eagle’s medium supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin under standard cell culture conditions (37 °C, 5% CO₂). Mononuclear cells from the bone marrow were isolated via density-gradient centrifugation using Ficoll-Paque PLUS solution. The interphase containing mononuclear cells was collected, washed, and cultured to allow the BMSCs to adhere. Once a sufficient number of BMSCs were obtained, the culture supernatant was collected and centrifuged at 2000 × g for 30 minutes at 4 °C to remove cells and debris. The supernatant was then filtered through a 0.45 μm filter to remove larger vesicles. Exosomes were pelleted by ultracentrifugation at 100000 × g for 70 minutes at 4 °C. The pellet was resuspended in phosphate-buffered saline (PBS), and this step was repeated to ensure purity. The final exosome pellet was resuspended in a minimal volume of PBS and stored at -80 °C for further analysis.

Characterization of exosomes

Exosome samples were initially adsorbed onto copper grids and stained with uranyl acetate to verify the identity, purity, and characteristics of the extracted exosomes. Subsequently, their morphology and size were evaluated via a transmission electron microscopy to confirm the typical “cup-shaped” appearance associated with exosomes. The size distribution and concentration of the exosomes were then determined using nanoparticle tracking analysis, providing a quantitative assessment of the exosome populations within the sample. Furthermore, western blotting or immunofluorescence assays employing specific antibodies were employed to identify surface markers, such as CD9 and CD63, on exosomes by confirming their origin as vesicles derived from exosomal sources. Additionally, fluorescent dyes (e.g., PKH67) have been used to label exosomes and track their uptake by target cells. This facilitated visualization under a confocal microscope and quantification using flow cytometry to ensure efficient internalization into cellular entities.

Differential miRNA analysis of exosomes derived from BMSCs

To extract and quantify the total RNA from BMSC-derived exosomes, the exosome pellet was resuspended in RNase-free water, and an appropriate volume of RNA lysis buffer was added to facilitate membrane disruption and RNA release. Following a 5-minute incubation at room temperature, chloroform was added to the mixture, which was then vortexed and centrifuged to separate the phases. The upper aqueous phase, enriched with RNA, was carefully transferred to a new tube and isopropanol was added to precipitate the RNA. After centrifugation, the supernatant was removed and the RNA pellet was washed with 75% ethanol. The dried RNA pellet was resuspended in RNase-free water and its concentration and purity were determined using a spectrophotometer. RNA integrity was assessed by agarose gel electrophoresis, confirming the suitability of the extracted RNA for downstream applications, such as gene expression analysis and miRNA profiling. All steps were conducted in an RNase-free environment using RNase-free reagents to prevent contamination and ensure the reliability of the results.

Following the ligation of the extracted RNA with 5’ and 3’ adapters, cDNA reverse transcription was performed to serve as a template for constructing the miRNA library. The resulting library was subjected to PE150 sequencing to generate sequence data. To identify the differentially expressed miRNAs in BMSC-derived exosomes, raw sequencing reads were quality-checked and trimmed using FastQC and Trimmomatic to remove low-quality reads and adapter sequences, respectively. Subsequently, the cleaned reads were aligned to the reference genome using Bowtie software, allowing precise mapping of miRNA sequences. The known miRNAs were annotated by comparing their aligned sequences with those in the miRBase database, which is a comprehensive repository of miRNA sequences. The miRDeep2 software was used to identify these known miRNAs and predict novel miRNAs by assessing the secondary structure of the precursor miRNAs and their alignment to the reference genome. The prediction of novel miRNAs involves the evaluation of the presence of a stable hairpin structure and the production of a mature miRNA sequence from the precursor.

DE-miRNAs were selected based on stringent criteria of P value < 0.05 and |log2 fold change| ≥ 1, ensuring the identification of miRNAs with substantial expression changes. Furthermore, ten differentially expressed miRNAs (DE-miRNAs) were carefully selected from both the upregulated and downregulated groups while considering the relevant literature pertaining to neuroinflammation. Five differentially regulated miRNAs from each group were selected for reverse transcription-polymerase chain reaction (RT-PCR) and quantitative PCR (qPCR) validation.

Exosome uptake assay

According to the manufacturer’s agreement, the PKH67 green fluorescent labeling kit (Umibio, Shanghai, China) was utilized for labeling the isolated exosomes from normal and epileptic mouse BMSCs. Briefly, exosomes were incubated with PKH67 dye for 10 minutes, followed by three washes to remove excess dye using OptimaMAX-XP (Backman, UT, United States). Subsequently, these cells were co-cultured with HT22 cells for 24 hours. After three additional washes with PBS, red CellLINK555 dye (Tianjiu Regeneration, Tianjin, China) was added and the cells were incubated for 30 minutes. After another round of washing with PBS, fixation was performed at room temperature with 4% paraformaldehyde for 30 minutes. The cells were then washed three times with PBS and stained with the nuclear stain DAPI for 10 minutes. The uptake of exosomes derived from normal and epileptic mouse BMSCs by HT22 cells was visualized through confocal microscopy analysis. The negative control group consisted of HT22 cells treated similarly to the other groups, but without the addition of PKH67 dye during co-incubation.

Functional experiments of exosome

This study investigated the functional effects of exosomal miR-203 on neuroinflammation in a cellular TLE model. HT22 cells were co-cultured with exosomes isolated from control group or TLE group BMSCs at a concentration of 10 μg/mL. Following 24-hour incubation, the expression levels of the target genes SOCS3 and miR-203-3p were quantified using qPCR. Moreover, changes in inflammatory factors (TNF-α, IL-6) in the cell supernatant were determined by enzyme-linked immunosorbent assay (ELISA). The extent of cellular apoptosis was evaluated using flow cytometry, and cell proliferation was assessed using the CCK8 assay.

Dual luciferase reporter assay

To validate the regulatory association between miR-203-3p and its target gene, SOCS3, we transfected HEK-293T cells with pmirGLO plasmids harboring wild-type and mutant SOCS3 promoter regions in conjunction with the overexpression of either miR-203-3p mimic (NC) or miR-203-3p. Following a 48-hour transfection period, the luminescence intensity of the firefly luciferase protein was quantified.

NF-κB pathway modulation study

To investigate the regulatory role of miR-203/SOCS3 in neuroinflammation through the NF-κB pathway in mice, we established an epilepsy cell model using HT22 cells. First, we determined the glutamate concentration required to induce HT22 epilepsy using a CCK8 assay. Subsequently, in the HT22 epilepsy cell model, we separately added miR-203-3p mimic or inhibitor, miR-203-3p inhibitor alone, and si-SOCS3/NC along with an NF-κB inhibitor (100 μmol/L)[28,29]. The RNA expression levels of miR-203-3p and its target gene, SOCS3, were assessed using qPCR. Additionally, western blotting technique was employed to detect the miRNA target gene (SOCS3, 1:2000, Proteintech, IL, United States) as well as markers of the NF-κB pathway: Inhibitor of kappa B kinase alpha/beta (1:1000, ZenBio, NC, United States), phospho-inhibitor of kappa B kinase alpha/beta (1:1000, ZenBio, NC, United States), NF-κB inhibitor alpha (1:10000, Proteintech, IL, United States), phospho-NF-κB inhibitor alpha (1:1000, ZenBio, NC, United States), p65 (1:10000, Proteintech, IL, United States) and p-p65 (1:1000, ZenBio, NC, United States). The secondary antibody was goat anti-rabbit horseradish peroxidase (Bioss, MA, United States) at a dilution of 1:5000. Cell apoptosis was evaluated, and cell proliferation was assessed using the CCK8 assay. Furthermore, inflammatory factors (TNF-α and IL-6) in the cell supernatant were quantified using ELISA.

Statistical analysis

Data were analyzed using GraphPad Prism 9 (Version 9.4.0) and presented as means ± SD. Statistical differences between groups were evaluated using the t-test or one-way ANOVA, with P < 0.05 considered statistically significant.

RESULTS

Examination of exosomes derived from mouse BMSCs

Transmission electron microscopy imaging revealed that exosomes from both the epilepsy and normal groups displayed the characteristic “cup-shape” morphology, with a clear membrane structure (Figure 1A). The average particle size and concentration were determined to be 85.31 nm and 1.62E+10 particles/mL for the epilepsy group, and 84.64 nm and 3.29E+10 particles/mL for the normal group, respectively (Figure 1B). Exosomes from both groups showed positive expression of the protein markers CD9 and CD63, with the epilepsy group showing a slightly higher positivity rate for CD9 (2.7%) than the normal group (2.1%). The expression of CD63 was minimal in both groups, with 0.6% and 0.3% positivity in the epilepsy and control groups, respectively (Figure 1C).

Figure 1 Characteristic of exosomes from bone marrow mesenchymal stem cells. A: Transmission electron microscopy images reveal the characteristic “cup-shaped” morphology and clear membrane structure of exosomes from both the epilepsy and normal groups of mice; B: Nanoparticle tracking analysis shows the average particle size and concentration to be 8531 nm and 1.62E+10 particles/mL for the epilepsy group, and 84.64 nm and 3.29E+10 particles/mL for the normal group, respectively; C: Flow cytometry analysis demonstrates positive expression of the protein markers CD9 and CD63 in exosomes from both groups, with a slightly higher positive rate for CD9 in the epilepsy group than in the normal group and minimal expression of CD63 in both groups. TLE: Temporal lobe epilepsy.

Identification and validation of differentially expressed miRNAs in epileptic mouse BMSCs

Table 1 presents the 20 miRNAs with the most significantly differential expression related to neuroinflammation, selected based on the degree of difference. This includes 10 miRNAs with upregulated expression and 10 with downregulated expression. A total of 10 differentially expressed miRNAs were selected for experimental validation by RT-qPCR, including five upregulated miRNAs (miR-144-3p, miR-193a-5p, miR-205-5p, miR-200a-3p, and miR-203-3p) and five downregulated miRNAs (miR-128-3p, miR-181c-5p, miR-23a-3p, miR-30b-3p, and miR-221-3p). Compared to extracellular vesicles from normal mouse BMSCs, the levels of extracellular vesicles in epileptic mouse BMSCs were significantly increased (P < 0.05), including an increase in the aforementioned upregulated miRNAs: MiR-144-3P, miR-193a-5P, miR-200a-3P, and miR-203–3P, whereas the levels of certain downregulated miRNAs decreased: MiR-181c-5P, miR-23a-3P, and miR-30b-3P (P < 0.05, Figure 2). However, no significant differences were observed between extracellular vesicles isolated from epileptic and normal mice for miR-205-5P, miR-128-3P, and miR-221-3P (P > 0.05, Figure 2). The consistency rate between the sequencing analysis results and RT-qPCR results was approximately 70%, indicating the high reliability of the sequencing results. Furthermore, owing to its more pronounced upregulation compared to miR-144-3p and miR-200a–3p, the focus of subsequent experiments was on miR-203-3p. qPCR validation of ten differentially expressed miRNAs associated with neuroinflammation is presented.

Figure 2 Quantitative polymerase chain reaction validation of DE-miRNAs. A-J: Expression miRNAs in epileptic mouse bone marrow mesenchymal stem cells. ^aP < 0.05, ^bP < 0.01, ^cP < 0.001, NS: No significance. TLE: Temporal lobe epilepsy.

Table 1 Twenty DE-miRNA associated with neuroinflammation.

MiRNA	Log2FC	P value	Padj value
mmu-miR-193a-5p	7.255065725	1.63E-10	1.44E-09
mmu-miR-205-5p	6.611947124	5.89E-33	1.63E-31
mmu-miR-203-3p	6.322996495	6.36E-07	4.34E-06
mmu-miR-200a-5p	5.553009313	0.004701826	0.020589048
mmu-miR-200a-3p	5.551982625	0.00094398	0.004619947
mmu-miR-182-5p	3.510594588	0.000514058	0.0026731
mmu-miR-200c-3p	3.372596465	3.08E-41	1.16E-39
mmu-miR-196a-5p	3.326742762	4.44E-13	4.62E-12
mmu-miR-196a-5p	3.291829303	8.00E-11	7.23E-10
mmu-miR-144-3p	3.077271591	1.10E-22	2.08E-21
mmu-miR-3473h-5p	-2.501671763	0.009780829	0.039890438
mmu-miR-700-3p	-2.477319455	0.000489188	0.002575975
mmu-miR-484	-2.343847305	7.95E-14	8.94E-13
mmu-miR-339-5p	-2.257736292	1.86E-19	3.23E-18
mmu-miR-328-3p	-2.212306543	5.48E-30	1.27E-28
mmu-miR-30b-5p	-2.151354017	0.000281059	0.00153843
mmu-miR-23a-3p	-2.129117956	4.09E-67	1.89E-65
mmu-miR-128-3p	-2.122702882	5.11E-20	9.25E-19
mmu-miR-181c-5p	-2.02817	7.15E-06	4.65E-05
mmu-miR-221-3p	-1.25811	0.007661	0.031871

MiRNA: MicroRNA; FC: Fold change.

Exosomes secreted by BMSCs enhance the secretion of inflammatory mediators in hippocampal neurons

After incubation with PKH67-labeled exosomes, HT22 cells showed significant fluorescence, indicating the efficient uptake of exosomes by the cells (Figure 3A). HT22 cells also showed significant fluorescence (Figure 3B), and incubation with FAM-labeled miR-203-3p from BMSCs (Figure 3C and D). This indicates the efficient uptake of miR-203-3p from exosomes by cells. The qPCR results revealed a significant increase in miR-203-3p levels (P = 0.0497, P < 0.05; Figure 3E) and a significant decrease in SOCS3 mRNA expression (P = 0.0217, P < 0.05; Figure 3F) in the TLE group. Flow cytometry analysis demonstrated an elevated apoptosis rate (P = 0.0005, P < 0.05, Figure 3G-K), while the CCK8 assay indicated a reduced cell proliferation ability (P = 0.00334, P < 0.05, Figure 3L). Furthermore, ELISA detection showed upregulated levels of inflammatory factors such as TNF-α and IL-6 in the TLE group (TNF-α: P = 0.0005, P < 0.05, Figure 3M; P = 0.0004, P < 0.05, Figure 3N).

Figure 3 Exosomes secreted by bone marrow mesenchymal stem cells enhance the secretion of inflammatory mediators in hippocampal neurons. A: Confocal microscopy images of HT22 cells co-cultured with bone marrow mesenchymal stem cell-derived exosomes. Cell nuclei were stained with DAPI, cell membranes were stained with CellLINK555, and exosomes were labeled with PKH67. The scale is 25 μm; B: Confocal microscopy imaging was performed after co-culturing HT22 cells with extracellular vesicles from bone marrow mesenchymal stem cells. DAPI staining visualized the cell nuclei, PKH26 staining labeled the cell membrane, and FAM labeling detected miR-203-3p within the extracellular vesicles. The scale bar represents 25 μm; C: Flow cytometry analysis of extracellular vesicles in the supernatant after HT22 cell transfection with FAM-miR-203-3p; D: Electron microscopic examination of extracellular vesicles in the supernatant after HT22 cell transfection with FAM-miR-203-3p; E: Quantitative polymerase chain reaction results reveal a significant increase in miR-203-3p levels in the temporal lobe epilepsy (TLE) group; F: Significant decrease in suppression of cytokine signaling 3 mRNA expression was observed in the TLE group; G-K: Flow cytometric analysis demonstrated an elevated apoptosis rate in the TLE group; L: CCK8 assay indicated reduced cell proliferation in the TLE group; M and N: Enzyme-linked immunosorbent assay detection shows upregulated levels of inflammatory factors such as tumor necrosis factor-α and interleukin-6 in the TLE group. ^bP < 0.01. TLE: Temporal lobe epilepsy; PBS: Phosphate-buffered saline; TNF-α: Tumor necrosis factor-α; IL-6: Interleukin-6.

MiR-203-3p/SOCS3 regulates neuroinflammation of hippocampal neurons in epileptic mice through the NF-κB pathway

In the presence of wild-type plasmid SOCS3 co-expressed with miR-203-3p or mimic NC, overexpression of miR-203-3p led to a significant reduction in firefly luciferase intensity (P = 0.0002, P < 0.05, Figure 4A). However, mutation of the binding site of SOCS3 restored firefly luciferase intensity. The IC₅₀ of glutamate in HT22 cells was determined to be 25.01 mmol/L using CCK8 assay (Figure 4B). In subsequent experiments, when this concentration was used as the induction dose for an epilepsy model, the expression level of miR-203-3p remained consistently higher than that in the untreated group, regardless of whether it was overexpressed by miR-203-3p mimics or suppressed by inhibitor treatment. Notably, miR-203-3p expression was significantly higher in the miR-203-3p mimic overexpression group than in the inhibitor group (Figure 4C). Although minimal changes were observed at the RNA level for the SOCS3 gene (Figure 4D), its protein level showed an overall decrease and was lower in the miR2033p mimics group than in the inhibitor group. Additionally, activation of NF-κB pathway markers along with increased levels of inflammation (IL-6 and TNF-α) were observed in the miR-203-3p mimics group (Figure 4E-N).

Figure 4 MiR-203-3p regulates inflammation by targeting the suppression of cytokine signaling 3-mediated nuclear factor kappaB pathway. A: Luciferase reporter assay showing the relative firefly luciferase activity in HEK-293T cells co-transfected with wild-type or mutant suppression of cytokine signaling 3 3’ untranslated region reporter plasmids and miR-203-3p mimic or miR-203-3p; B: CCK8 assay to determine the IC₅₀ concentration of glutamate required to induce HT22 epilepsy; C: Quantitative polymerase chain reaction analysis of miR-203-3p expression in HT22 cells after treatment with miR-203-3p mimic or inhibitor; D: Quantitative polymerase chain reaction analysis of suppression of cytokine signaling 3 mRNA expression in HT22 cells under the conditions described in Figure 3C; E-L: Western blot analysis of key proteins involved in the nuclear factor kappaB pathway, including total and phosphorylated forms of inhibitor of kappa B kinase alpha/beta, nuclear factor kappaB inhibitor alpha, and p65, in HT22 cells treated with miR-203-3p mimics or inhibitors; M and N: Enzyme-linked immunosorbent assay measurements of the inflammatory cytokines interleukin-6 and tumor necrosis factor-α in the supernatant of HT22 cells treated as in Figure 3C. Data are presented as mean ± SEM of three independent experiments. ^aP < 0.05, ^bP < 0.01, ^cP < 0.001. SOCS3: Suppression of cytokine signaling 3; IKKα/β: Inhibitor of kappa B kinase alpha/beta; p-IKKα/β: Phospho-inhibitor of kappa B kinase alpha/beta; IKBα: Nuclear factor kappaB inhibitor alpha; p-IKBα: Phospho-nuclear factor kappaB inhibitor alpha; TNF-α: Tumor necrosis factor-α; IL-6: Interleukin-6.

Compared with the single knockout of miR-203 3p in the HT22 epileptic cell model, the double-knockout group of miR-203-3p and SOCS3 showed no difference in miR-203-3p expression (Figure 5A), while the expression levels of SOCS3 RNA (Figure 5B) and protein (Figure 5C and D) decreased. Furthermore, the double knockout group also demonstrated restoration of NF-κB pathway markers (Figure 5E-J), as well as IL-6 (Figure 5K) and TNF-α levels (Figure 5L). Additionally, after knocking out SOCS3 in the HT22 epileptic cell model, no significant difference was observed in the expression of miR-203-3p (Figure 6A), while the expression of SOCS3 RNA and protein was significantly reduced (Figure 6B-D). Moreover, an up-regulation of markers related to the NF-κB pathway was observed. However, upon administration of an inhibitor targeting the NF-κB pathway, although markers associated with the NF-κB pathway still exhibited upregulation (Figure 6E-J), a concomitant reduction in IL-6 and TNF-α levels was observed (Figure 6K and L).

Figure 5 Downregulation of miR-203-3p and suppression of cytokine signaling 3 can reinstate an inflammatory phenotype. A: Schematic representation of the experimental design of HT22 cells co-transfected with the miR-203-3p inhibitor and si-suppression of cytokine signaling 3 (SOCS3) or si-NC; B: Quantitative polymerase chain reaction analysis of SOCS3 mRNA expression in HT22 cells following co-transfection; C and D: Western blot analysis of SOCS3 protein levels in HT22 cells under the same conditions as Figure 5C; E-J: Western blot analysis of nuclear factor kappaB pathway markers in HT22 cells co-transfected with miR-203-3p inhibitor and si-SOCS3 or si-NC; K and L: Enzyme-linked immunosorbent assay measurements of interleukin-6 and tumor necrosis factor-α levels in the supernatant of HT22 cells co-transfected as in Figure 5C. Data are presented as mean ± SEM of three independent experiments. ^aP < 0.05, ^bP < 0.01, ^cP < 0.001, NS: No significance. SOCS3: Suppression of cytokine signaling 3; IKKα/β: Inhibitor of kappa B kinase alpha/beta; p-IKKα/β: Phospho-inhibitor of kappa B kinase alpha/beta; IKBα: Nuclear factor kappaB inhibitor alpha; p-IKBα: Phospho-nuclear factor kappaB inhibitor alpha; TNF-α: Tumor necrosis factor-α; IL-6: Interleukin-6.

Figure 6 Inhibition of the target gene suppression of cytokine signaling 3 can activate the nuclear factor kappaB pathway and promote neuroinflammation. A: Schematic representation of the experimental design for HT22 cells transfected with si-suppression of cytokine signaling 3 (SOCS3) or si-NC, with or without nuclear factor kappaB pathway inhibitor treatment; B: Quantitative polymerase chain reaction analysis of SOCS3 mRNA expression in HT22 cells after transfection; C-J: Western blot analysis of SOCS3 protein levels and nuclear factor kappaB pathway markers in HT22 cells under the same conditions as Figure 6C; K and L: Enzyme-linked immunosorbent assay measurements of interleukin-6 and tumor necrosis factor-α levels in the supernatant of HT22 cells treated as in Figure 6C. Data are presented as mean ± SEM of three independent experiments. ^aP < 0.05, ^bP < 0.01, ^cP < 0.001, NS: No significance. SOCS3: Suppression of cytokine signaling 3; NF-κB: Nuclear factor kappaB; IKKα/β: Inhibitor of kappa B kinase alpha/beta; p-IKKα/β: Phospho-inhibitor of kappa B kinase alpha/beta; IKBα: Nuclear factor kappaB inhibitor alpha; p-IKBα: Phospho-nuclear factor kappaB inhibitor alpha; TNF-α: Tumor necrosis factor-α; IL-6: Interleukin-6.

Exosomes regulate the expression of SOCS3 in the hippocampus through miR-203, affecting epileptic seizures

In kainic acid-induced TLE mice, both plasma and hippocampal levels of miR-203-3p were significantly upregulated (Figure 7A and B), while only the hippocampal expression of SOCS3 was decreased (Figure 7C). Importantly, no significant difference was observed in the plasma levels of SOCS3 compared to those in the control group (Figure 7D).

Figure 7 Differences between temporal lobe epilepsy mouse plasma and the expression of miR-203-3p and suppression of cytokine signaling 3 in the hippocampal region. A-D: Quantitative polymerase chain reaction analysis of miR-203-3p and suppression of cytokine signaling 3 mRNA expression in the plasma and hippocampal regions of the temporal lobe epilepsy model. ^aP < 0.05, ^cP < 0.001. TLE: Temporal lobe epilepsy.

DISCUSSION

Our study revealed significant upregulation of exosomal miR-203 in BMSCs derived from epileptic mice. This finding suggests a potential role for miR-203 in the pathogenesis of TLE. The increased levels of miR-203 in exosomes from epileptic mice may be a response to ongoing neuroinflammatory processes in the brain. However, the role of miR-203 in epilepsy is poorly understood. Our findings indicated that miR-203 may serve as a critical regulator of the neuroinflammatory response associated with TLE. Similar to the work of Wang et al[15], circulating exosomal miRNAs have been identified as potential biomarkers in the diagnosis and treatment of DRE. The differential expression of miR-203 in BMSC-derived exosomes from epileptic mice compared to controls highlights its potential as a novel biomarker for epilepsy, a concept also supported by García-Gracia et al[14] and Jeppesen et al[30].

Our data demonstrate that exosomal miR-203 from BMSCs can be internalized by hippocampal neurons, leading to the downregulation of SOCS3 and subsequent activation of the NF-κB pathway. This pathway is well-known for its pro-inflammatory effects, which can exacerbate the neuroinflammatory state in TLE. This is in line with the findings of Xian et al[31], who showed that NF-κB activation is a key mediator of the neuroinflammatory processes of epilepsy. The enhanced secretion of pro-inflammatory cytokines, such as TNF-α and IL-6, following the uptake of exosomal miR-203 by hippocampal neurons, underscores the significant impact of neuroinflammation in the progression of epilepsy. This finding is consistent with the growing body of evidence that implicates neuroinflammation as a key factor in the pathophysiology of epilepsy[32,33].

Exosomes have emerged as important mediators of intercellular communication, particularly in neurodegenerative diseases. Our study adds to this body of knowledge by showing that exosomes can transfer miR-203 from BMSCs to neurons, thereby modulating the neuroinflammatory responses in epilepsy. This is supported by the work of Cui et al[34], who demonstrated the transfer of miRNAs via exosomes in a mouse model of Alzheimer’s disease. The modulate expression of miR-203 is a promising therapeutic strategy for epilepsy treatment. Targeting miR-203 could reduce the neuroinflammatory response and improve outcomes in patients with DRE, as suggested by the therapeutic potential of miRNAs discussed by Dixit et al[35] and Boileau et al[36].

Although previous studies have explored the role of miRNAs in epilepsy, the focus on miR-203 and its specific role in modulating the SOCS3/NF-κB pathway is novel. Our findings expand the current understanding of the miRNA-mediated regulation of epilepsy and highlight the potential of exosome-based therapies. This aligns with the innovative approach adopted by Wang et al[37] in their study of exosomal miRNAs in a stroke model. One limitation of our study is the use of a single animal model and the need for further validation in human studies. Future research should investigate the long-term effects of miR-203 modulation and explore the potential of exosomal miR-203 as a therapeutic agent in clinical trials, as recommended by Batrakova and Kim[38] in their review on the translational potential of exosome research. Another limitation is the lack of additional experiments to comprehensively analyze potential treatments and their safety. Although our study provides preliminary evidence of miR-203’s involvement in neuroinflammation, it does not fully explore the therapeutic effects of targeting miR-203. Future studies should include in vivo experiments to assess the efficacy and safety of miR-203-targeting therapies in TLE models. This could involve the administration of miR-203 inhibitors or the delivery of exosomes engineered to modulate miR-203 levels, followed by monitoring of seizure frequency, severity, and duration, as well as evaluating any potential side effects on cognitive and behavioral functions.

CONCLUSION

The clinical implications of our findings are significant as they provide a basis for developing new therapeutic strategies for epilepsy. Targeting exosomal miR-203 could offer a noninvasive approach to modulate neuroinflammation and improve patient outcomes, particularly in patients with DRE. This is supported by the clinical studies by Gonçalves et al[39], who highlighted the importance of non-invasive biomarkers in epilepsy management. In conclusion, our study identifies exosomal miR-203 as a key regulator of neuroinflammation in a mouse model of epilepsy. Targeting miR-203 may offer a novel therapeutic strategy for epilepsy by modulating the SOCS3/NF-κB pathway, thus providing a potential avenue for the development of cell-free therapeutics.