Table 1.
Histopathology grading system.
Fig 1.
Effects of L. gasseri ATCC33323 supplementation on DSS-induced colitis symptoms.
a. Study design. Villin-Cre;CDH1wt/flox mice were crossbred to serve as the experimental group, and CDH1wt/flox mice served as the control group. L. gasseri ATCC33323 or PBS was gavaged for 21 consecutive days, with simultaneous ad libitum access to 2.5% (w/v) DSS in the latter 7 days. b. PCR analysis of hybrid offspring between CDH1flox/flox mice and transgenic mice harboring Villin-Cre;CDH1: Mutant = 310 bp, Wild = 243 bp; Villin-Cre = 567 bp; c. DAI index during DSS modeling; d. Survival at DSS modeling; e. Percentage change in body weight during DSS modeling; f. Colon length; g. Detection of mouse colonic permeability via the FITC–dextran assay; h. Mouse colon tissue. The values are expressed as the means ± SDs (n = 8). Superscript letters indicate significant differences at *P < 0.05, **P < 0.01, and ***P < 0.001.
Fig 2.
Effects of L. gasseri ATCC33323 gavage on intestinal health, intestinal histopathology and inflammation during colitis induction.
a. Pathologic section of the mouse colon (H&E) at 200× magnification. 1. Inflammatory cell infiltration; 2. Disappearance of crypts; b. pathological scoring of colon severity; c. MPO activity; d. MDA activity; e. SOD activity; f. IL-1β, IL-6, and TNFα cytokines; g. Western blot validation of mucosal barrier protein expression; h. Statistical analysis of grayscale values of mucosal barrier protein western blot. The values are expressed as the means ± SDs (n = 8). Superscript letters indicate significant differences at *P < 0.05, **P < 0.01, and ***P < 0.001.
Fig 3.
Effects of L. gasseri ATCC33323 supplementation on the expression and localization of mucosal proteins in DSS-induced colitis.
Histopathological sections of the mouse colon were observed at 200× magnification. a. E-cadherin; b. MUC2; c. ZO-1; d. occludin; e. claudin1; f. Statistical analysis of immunohistochemical staining scores for mucosal barrier proteins.
Fig 4.
Effects of L. gasseri ATCC33323 supplementation on the expression and localization of the E-cadherin/β-catenin complex and the E-cadherin/p120-catenin complex in DSS-induced colitis; magnification, 400×.
a. Fluorescence staining of the E-cadherin/β-catenin complex; b. Statistical analysis of the average fluorescence intensity of the E-cadherin/β-catenin complex; c. Fluorescence staining of the E-cadherin/p120-catenin complex; d. Statistical analysis of the average fluorescence intensity of the E-cadherin/p120-catenin complex.
Fig 5.
16S rDNA sequencing and qPCR were used to determine the effects of L. gasseri ATCC33323 on the intestinal microbiota of the mice, and the fecal bacteria of six groups of mice were analyzed.
a. Alpha diversity analysis of the mouse gut microbiota, Chao1 index; b. Alpha diversity analysis of the mouse gut microbiota, Shannon index; c. Alpha diversity analysis of the mouse gut microbiota, Simpson index; d. Beta diversity analysis of the mouse gut microbiota, PCA; e. Beta diversity analysis of the mouse gut microbiota, PCoA; f. composition of the gut microbiota at the phylum level among each group; g. composition of the gut microbiota at the family level among each group; h. determination of Lactobacillus spp. gene copy number levels in the DNA of each group of mouse fecal samples by qPCR and analysis of amplified samples by agarose gel electrophoresis; i. determination of L. gasseri ATCC33323 gene copy number levels in the DNA of each group of mouse fecal samples by qPCR and analysis of amplified samples by agarose gel electrophoresis.
Fig 6.
RNA-seq analysis of colon tissue.
a. Volcano plots were used to analyze differential genes between the NC DSS and NC LAB groups; b. Volcano plots were used to analyze differential genes between the NC LAB and CDH1+/- LAB groups; c. Heatmaps were used to analyze the differential genes between the NC DSS, NC LAB, CDH1+/- DSS, and CDH1+/- LAB groups; d. GO enrichment analysis between the NC DSS and NC LAB groups; e. GO enrichment analysis between the NC LAB and CDH1+/- LAB groups; f. KEGG enrichment analysis between the NC DSS and NC LAB groups; g. KEGG enrichment analysis between the NC LAB and CDH1+/- LAB groups.
Fig 7.
In vitro study of L. gasseri ATCC33323 with colon cancer cell lines.
a. qPCR assay to analyze the differential gene expression of L. gasseri ATCC33323 after 3/6/12 h of coculture with HCT116 and Caco2 colon cancer cells; b. Western blot analysis of NR1I3 and E-cadherin expression in L. gasseri ATCC33323 after 3/6/12 h of coculture with HCT116 and Caco2 colon cancer cells; c. Validation of the knockdown efficiency of NR1I3 in HCT116 and Caco2 colon cancer cells; d. NR1I3 and E-cadherin expression after intracellular NR1I3 knockdown in coculture with L. gasseri ATCC33323 for 3/6/12 h; e.,f. Results of ChIP experiments showing that NR1I3 can act as a transcription factor for CDH1; g. Luciferase reporter gene assay to verify the activity of NR1I3 as a CDH1 transcription factor. The values are expressed as the means ± SDs (n = 3). Superscript letters indicate significant differences at *P < 0.05, **P < 0.01, and ***P < 0.001.
Fig 8.
Effects of L. gasseri ATCC33323 supplementation on LPS-stimulated cellular inflammatory responses.
a. E-cadherin expression in HCT116 cell lines with or without NR1I3 knockdown by L. gasseri ATCC33323 pretreatment; b. E-cadherin expression in Caco2 cell lines with or without NR1I3 knockdown by L. gasseri ATCC33323 pretreatment; c. IL-1β, IL-6 and TNFα expression in HCT116 cell lines with or without NR1I3 knockdown by L. gasseri ATCC33323 pretreatment; d. IL-1β, IL-6 and TNFα expression in Caco2 cell lines with or without NR1I3 knockdown by L. gasseri ATCC33323 pretreatment. The values are expressed as the means ± SDs (n = 3). Superscript letters indicate significant differences at *P < 0.05, **P < 0.01, and ***P < 0.001.