Fig 1: IL-13 preserves BBB integrity and attenuates pyroptosis in bEnd.3 cells after OGD/R injury. A Experimental workflow of OGD/R modeling and treatment in bEnd.3 cells. (B-C) IL-5 (B) and IL-13 (C) levels in supernatants of bEnd.3 cells after OGD/R ± IL-25 treatment (n = 6 per group). D-H Western blot analysis of bEnd.3 cells after OGD/R with or without IL-25, IL-5, or IL-13 treatment: Representative blots (D), ZO-1 quantification (E), Occludin quantification (F), Claudin-5 quantification (G), Caspase-1 quantification (H) (n = 6 per group). I-J Representative flow cytometry plots (I) and quantitative analysis (J) of apoptosis in bEnd.3 cells subjected to OGD/R with or without IL-25, IL-5, or IL-13 treatment (n = 6 per group). Viable cells: Annexin V⁻/PI⁻; Early apoptotic: Annexin V⁺/PI⁻; Late apoptotic: Annexin V⁺/PI⁺. K Immunofluorescence of ZO-1 and Claudin-5 (green) in bEnd.3 cells monolayers after OGD/R ± IL-13 treatment. Nuclei counterstained with DAPI (blue). Scale bar = 100 μm. Data presented as mean ± SD; ns: not significant; *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001
Fig 2: IL-13 improves BBB function by reducing CXCL-10 expression. A-B Cytokine array analysis identified CXCL-10 as one of the significantly downregulated cytokines in the IL-13-treated groups (n = 3 per group). C-D CXCL-10 expression in bEnd.3 cells subjected to OGD/R ± IL-25, IL-5, or IL-13: Representative Western blot (C) and quantification (D) (n = 6 per group). E-F CXCL-10 levels in ipsilateral cortex 3 days after TBI: Representative blot (E) and quantification (F) (n = 6 per group). G Experimental workflow of TBI modeling and therapeutic administration. H-K Western blot analysis of TJs (ZO-1, Occludin, Claudin-5) in the ipsilateral cortex 3 days after TBI: Representative blots (H), ZO-1 quantification (I), Occludin quantification (J), Claudin-5 quantification (K) (n = 6 per group). Data presented as mean ± SD; ns: not significant; *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001
Fig 3: IL-25 activates ILC2s and stimulates the production of downstream factors IL-5/IL-13. A Single-Cell transcriptomic atlas of after TBI mice brain UMAP projection of 29,046 single cells isolated from cerebral cortex tissues of Sham (7,963), TBI + PBS (9,332 ), and TBI + IL-25 (11,751) groups. Colors represent distinct cell populations identified through unsupervised clustering. B Proportions of BBB-associated cells (endothelial cells, astrocytes, pericytes) across Sham, TBI + PBS, and TBI + IL-25 groups. C Heatmap visualization of differentially expressed genes in endothelial cells isolated from Sham, TBI + PBS, and TBI + IL-25 treatment groups. D KEGG network pathway enrichment analysis of differentially expressed genes in endothelial cells from the SHAM, TBI + PBS, and TBI + IL-25 groups. E Integrated KEGG pathway analysis of the three groups. F Cell-cell communication analysis depicting differential gene expression between endothelial cells and ILC2s. G Flow cytometry gating strategy for ILC2 identification: lymphocytes were selected by FSC/SSC, followed by detection of Lin-CD45+CD127+ST2+ cells. H-I Representative flow plots (H) and quantitative analysis (I) of cerebral cortex ILC2s at 3 days after TBI (n = 3 per group). J-M Cytokine levels in ipsilateral cerebral cortex tissue and serum at 3 days after TBI: IL-5 (cerebral cortex) (J), IL-5 (serum) (K), IL-13 (cerebral cortex) (L), IL-13 (serum) (M) (n = 6 per group). Data presented as mean ± SD; ns: not significant; *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001
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