Fig 2: FLaAN/C attenuated NLRP3-mediated pyroptosis in EAE mice. (A) Abundance of caspase-1 p20, GSDMD-N, and IL-18 and IL-1ß proteins in mouse spinal cord. (B–C) The mRNA expression of Caspase-1 (B) and GSDMD (C) were quantitated by qRT-PCR. (D) Caspase-1 activity in mouse spinal cord was detected using the kit. (E) Detection of NLRP3 and ASC complex formation in spinal cord by co-immunoprecipitation. (F–G) Representative images of NLRP3+ (F) and ASC+ (G) in CD11b+ cells in sections of paraffin-embedded spinal cord tissue (scale bars: 50 µm). Data are shown as means ± SD (n = 6 per group). *p < 0.05, **p < 0.01, ***p < 0.001; # p < 0.05, ## p < 0.01, ### p < 0.001. * vs. Control group; # vs. MOG group.

Fig 3: Hypoxia-preconditioned OM-MSCs downregulated the level of molecules closely related to pyroptosis. (A) Western blotting results of NLRP3, ASC, cleaved caspase-1, cleaved caspase-8, GSDMD, and mature IL-1ß in the cortex of the perihematomal regions at 3 days post-ICH. (B) Quantitative analysis of target proteins. Data are expressed as the mean ± SEM (n=3) (**, P<0.01; ***, P<0.001). Abbreviations: OM-MSCs, olfactory mucosa mesenchymal stem cells; ICH, intracerebral hemorrhage; NLRP3, nod-like receptor family protein 3; ASC, apoptosis-associated speck-like proteins containing a caspase recruitment domain; GSDMD, Gasdermin D; SEM, standard error of mean.

Fig 4: Neuroinflammatory activity enhanced in the hippocampus in Cx3cr1CreERIL-10-/-mice. a Western blotting bands of inflammation-related molecules in littermates and Cx3cr1CreER IL-10-/-mice. Bar graphs represent densitometric plots of protein expression in littermate and Cx3cr1CreERIL-10-/- mice in GFAP (b), Iba1 (c), NLRP3 (d), caspase-1 (e), Pro-IL-1ß (f), IL-1ß (g), Pro-IL-18 (h), IL-18 (i), ASC (j), IL-10 (k), and IL-6 (l). Each dot represents a mouse. Bars represent mean ± SEM. n = 4 in each group. Significant differences were established by t-test. m Expression of GFAP and Iba1 in CA3 region in mice hippocampal slices. Immunofluorescence images, captured with a 10 × objective. Green, immunoreactivity of GFAP and Iba1; blue, nuclei staining with DAPI. The merged images of GFAP, Iba1 and DAPI staining. n Immunofluorescence co-localization of NLRP3 and Iba1 in CA3 region in mice hippocampal slices. Immunofluorescence images, captured with a 10 × objective. Green, immunoreactivity of NLRP3; Red, immunoreactivity of Iba1; Blue, nuclei staining with DAPI. The merged images of NLRP3, Iba1 and DAPI staining. *P < 0.05, **P < 0.01

Fig 5: Microglial activation in the hippocampus following myocardial infarction.(a) Shown are representative immunofluorescence images of hippocampal cornu ammonis (CA1) microglia stained for ionised calcium-binding adapter molecule-1 (Iba-1); Iba-1 positive microglia were classified as ramified (top), intermediate (bottom), or active (not shown) based on their morphology in CA1 and dentate gyrus (DG) hippocampus regions. Myocardial infarction (MI) decreased the proportion of ramified microglia and increased the proportion of intermediate and active microglia (n = 8 sections from N = 4 mice). MI increases (b) interleukin-1ß (IL-1ß; N = 9) and (c) interleukin-18 (IL-18; N = 9) mRNA expression in the hippocampus; (d) hippocampal IL-1 receptor (IL-1R) protein expression is also increased following MI (N = 9). Panels e and f display representative immunofluorescence images of CA1 hippocampal microglia co-stained for Iba-1 (white) and (e) CD68 (red) or (f) apoptosis-associated speck-like protein containing a caspase-activation and recruitment domain (ASC; red); low (top) and high (bottom) abundance CD68 and ASC signals are shown. MI increases the proportion of (e) CD68-positive microglia (n = 8 sections from N = 4) and (f) ASC-positive microglia (n = 7 sections from N = 4) in CA1 and DG hippocampus regions. * denotes p < 0.05 for unpaired comparisons. Panels a, b, d, e, and f are presented as mean ± SEM and are compared with Student's t-test; Panel c is presented as median ± interquartile range and is compared with a Mann–Whitney test. Bar over micrographs is 50 µm in Panels a and e and 10 µm in Panel f.