Fig 1: CRE reduces the neuroinflammatory state induced by lipopolysaccharide. (A) The experiment outlines the timetable of the animal experiments. The mice were treated with CRE extract orally for 25 d, and the control group was administered distilled water of the same volume as the CRE extract. lipopolysaccharide treatment was given on day 19 at 750 μg/kg/body weight for a week. The mice were sacrificed before 8 h lipopolysaccharide treatment at 1.5 mg/kg body weight, and their organs were collected. (B) The changes in mouse body weight on days 0, 19, and 25. The statistical analysis was performed using one-way analysis of variance (ANOVA) and Tukey post hoc analysis. The results are presented as means ± SD, with * p < 0.05 and ** p < 0.01 indicating significance compared to the lipopolysaccharide group. (C) Serum IL-1β levels were measured, with the lipopolysaccharide group showing a significant increase compared to the control group and the lipopolysaccharide + CRE 50 mg/kg and lipopolysaccharide + CRE 100 mg/kg groups showing a significant decrease compared to the lipopolysaccharide group. (D) Il-1b mRNA levels were measured in the cerebrum and hippocampus of each group of male mice. (E) GFAP were evaluated in the cerebrum of each group of male mice by Western blot analysis. α-Tubulin was used as an internal control. (F) GFAP were evaluated in the hippocampus of each group of male mice. α-Tubulin was used for internal control. The statistical analysis was performed using one-way ANOVA and Tukey post hoc analysis. The values represent means ± SD # p < 0.001 indicating significance compared to the control group and ** p < 0.01, *** p < 0.001 indicating significance compared to the lipopolysaccharide group.
Fig 2: Increased IL-1 signals enhance the mucin secretion in human colitis samples.(A) Box plots of mRNA levels of GLI1 and ILs in healthy and colitis specimens (using datasets GSE53306 and GSE38713). In the box plots, the middle line depicts the median and the whiskers in the min-to-max range. UC, ulcerative colitis. (B) The positive correlation between GLI1 and IL-1A or IL-1B in human datasets GSE53306 and GSE38713. (C) Representative images of H&E staining in human large intestine and colitis sections. The red lines show the column epithelial cell structure and the blue lines show the squamous epithelial cell structure in colitis model. Scale bars, 100 μm. (D) Gli1 and Ki67 immunostaining and quantification of human colon derived from healthy and ulcerative colitis specimens. Scale bars, 100 μm. (E) IL-1α and Gli1 immunostaining and quantification of human colon derived from healthy and ulcerative colitis specimens. Scale bars, 100 μm. (F) qRT-PCR analysis of mucin genes in human large intestinal organoids after treated with 10 nM IL-1 or IL-17 for 3 days. (G) Representative images of Muc2 and E-cadherin immunofluorescence costaining of human colon organoids after treated with IL-1 or IL-17 for 3 days. Scale bars, 100 μm. (H) A working model shows that disruption of BMP signaling in Gli1+ stromal cells increases goblet cell number and promotes mucin production through IL-1 and IL-17. *P < 0.05, **P < 0.01, and ***P < 0.001 by one-way ANOVA test in (D) and (E) and two-way ANOVA followed by Tukey’s multiple comparisons test in (F).
Fig 3: IL-1 and IL-17 promote goblet cell differentiation and enhance mucin production in mouse colonic organoids through NF-κB signaling.(A) Representative images of Muc2-mCherry-labeled distal colon organoids at day 3 after the treatments with 2 μM IWP-2, 10 μM DAPT, or 10 nM ILs. Scale bars, 100 μm. (B) FACS analysis and quantitation of Muc2-mCherry+ cells from distal colon organoids at day 3 after the treatments with 2 μM IWP-2, 10 μM DAPT, or 10 nM ILs. (C) qRT-PCR analysis of epithelial marker genes in distal colon organoids at day 3 after the treatments with 2 μM IWP-2, 10 μM DAPT, or 10 nM ILs. (D) qRT-PCR analysis of mucin genes in distal colon organoids cocultured with Gli1-tdTomato+ cells from indicated mice and treated with 50 nM IL-1Ra for 3 days. (E) Heatmap of the up-regulated genes in distal colon organoids after the treatments with 10 nM ILs for 12 hours. (F) Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis for the up-regulated signaling pathways after the indicated treatments for 12 hours. (G) Immunoblot analysis of the expression level of IL-17 in distal colon organoids treated with 10 nM ILs for 3 days. (H) qRT-PCR analysis of mucin genes in distal colon organoids after the indicated treatments for 3 days. *P < 0.05, **P < 0.01, and ***P < 0.001 by one-way ANOVA test in (B) and two-way ANOVA followed by Tukey’s multiple comparisons test in (C), (D), and (H).
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