Adipose-derived stem cell exosomes suppress NLRP3-mediated neuronal pyroptosis to attenuate seizures in a kainic acid-induced temporal lobe epilepsy model

Figures

• 

  Figure 1. Schematic illustration of experimental setup and timeline. After KA-induced epilepsy modeling in mice, seizure severity was assessed via 24-hour behavioral observation. On day 7 post-modeling, epidural electrodes were implanted with a 7-day recovery period. Continuous 7-day EEG-video monitoring was performed from days 14–21. Adipose-derived stem cell exosomes were administered intravenously on days 1, 7, and 14. On day 21, mice were euthanized for sample collection.

• 

  Figure 2. Characterization of ADSCs and exosomes. (A) Collection of adipose tissue obtained immediately by liposuction. (B) Adipose tissue after oil and water removal. (C) Representative image showing the typical spindle-like shape of isolated ADSCs under a light microscope. (D) Representative images of ADSCs differentiated into chondrocytes, osteogenic cells, and adipocytes, stained with Alcian Blue (a), Alizarin Red S (b), and Oil Red O (c), respectively. (E) Flow cytometry showed that CD29, CD44 and CD90 was positive and CD31, or CD45 was negative among the surface markers ADSCs. (F) Transmission electron microscopy (TEM) image of ADSCs derived exosomes revealing cup-shaped morphology (black arrows). (G) Nanoparticle tracking analysis (NTA) showing the size distribution of ADSCs derived exosomes. (H) Western blot analysis of CD63, TSG101, CD9, calnexin in ADSC-Exos and ADSCs (n = 3 per group).

• 

  Figure 3. PKH26 labeling to depict ADSC-Exos in brain parenchyma. (A) Representative image shows that PKH26-labeled ADSC-Exos cross the blood-brain barrier and accumulate in brain tissue over time. Fluorescence images show PKH26-labeled exosomes (red) in brain tissue at 2, 4, and 6 h post-injection (left to right). Nuclei were stained with Hoechst (blue). Strongest exosome accumulation was observed at 4 (h) (B) Quantitative analysis of the relative expression of PKH26-labeled ADSC-Exos in Hippocampus. Individual data points represent values from each mouse (n=3 per group). Data are expressed as mean ± SD. *p < 0.05, **p < 0.01(one-way ANOVA with Tukey’s post hoc test).

• 

  Figure 4. Therapeutic effects of ADSC-Exos on seizure activity in KA-induced temporal lobe epilepsy (TLE) mice. (A) Stereotaxic intrahippocampal KA injection (coordinates: AP −2.0 mm, ML −1.8 mm [right hemisphere], DV −1.8 mm). (B) Intravenous administration of ADSC-Exos via tail vein. (C) Surgical implantation of epidural EEG electrodes. (D–F) Quantitative analyses of (D) average seizure scores on Day 1 post-KA injection, (E) frequency of SRSs during the chronic phase (days 14–21), and (F) mean duration of spontaneous recurrent seizures (SRSs). Individual data points represent values from each mouse (n=5 per group). Data are expressed as mean ± SD. Statistical significance: *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001(one-way ANOVA with Tukey’s post hoc test). (G) Representative 1-minute EEG traces during epileptic episodes.

• 

  Figure 5. Effects of ADSC-Exos on KA-induced hippocampal neuron damage. (A) Representative Nissl-stained images (200×) showing ADSC-Exos attenuated KA-induced neuron loss in hippocampal CA1 and CA3 regions. (B) Representative TUNEL-stained images (200×) showing ADSC-Exos alleviated KA-induced apoptosis in CA1 and CA3. (C–F) Quantitative analyses of (C) CA1 Nissl+, (D) CA3 Nissl+, (E) CA1 TUNEL+, and (F) CA3 TUNEL+ cells. Individual data points represent values from each mouse (n=5 per group). Data are expressed as mean ± SD. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 (one-way ANOVA with Tukey’s post hoc test).

• 

  Figure 6. ADSC-Exos mitigate NLRP3-mediated pyroptosis in KA-induced temporal lobe epilepsy. (A–F) Western blot analysis and quantification of (A) representative immunoblots and protein levels of (B) NLRP3, (C) GSDMD-FL, (D) GSDMD-N, (E) Caspase-1, and (F) IL-1β in the Control, KA, KA+ADSC-Exos, and KA+Nig+ADSC-Exos groups. (G–J) qRT-PCR analysis of relative mRNA expression of (G) NLRP3, (H) GSDMD, (I) Caspase-1, and (J) IL-1β. (K–L) ELISA quantification of (K) serum IL-1β and (L) IL-18 levels. Individual data points represent values from each mouse (n=5 per group). Data are expressed as mean ± SD. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 (one-way ANOVA with Tukey’s post hoc test).

• 

  Figure 7. Bioinformatics analysis of hippocampal miRNA profiles from patients with refractory epilepsy (GSE99455, GEO database) and ADSC-derived exosomal miRNAs (GSE281527, GEO database). (A) Heatmap showing the top 50 most abundant miRNAs in ADSC-Exos. (B) Heatmap of 199 significantly downregulated hippocampal miRNAs (|log2FC| > 1, adj. p < 0.05) in epileptic patients versus healthy controls. (C) Volcano plot of differentially expressed miRNAs in the epileptic hippocampus. (D) Venn diagram showing 20 shared miRNAs that were enriched in ADSC-Exos and downregulated in epileptic hippocampi. (E–I) miRNA–target interaction analyses of 20 key miRNAs (hsa-mir-1587, hsa-mir-184, hsa-mir-30b-3p, hsa-mir-320b, hsa-mir-320e, hsa-mir-378a-3p, hsa-mir-4259, hsa-mir-4419b, hsa-mir-4459, hsa-mir-4660, hsa-mir-4768-5p, hsa-mir-504-3p, hsa-mir-539-5p, hsa-mir-584-5p, hsa-mir-6131, hsa-mir-616-3p, hsa-mir-629-5p, hsa-mir-6818-5p, hsa-mir-6826-5p, and hsa-mir-7-5p): (E) Heatmap showing differential expression of key miRNA–target pairs. (F) Sankey diagram illustrating miRNA–gene–pathway relationships. (G) Network visualization of core miRNA–target interactions. (H) Bubble plot ranked by target genes (y-axis:…

• 

  Figure 8. A schematic representation illustrates mechanisms by which adipose-derived stem cell exosomes suppress NLRP3-mediated pyroptosis to alleviate temporal lobe epilepsy.