Portulaca oleracea L.-derived exosome-like nanoparticles ameliorate colitis by modulating gut microbiota and metabolites to reduce endoplasmic reticulum stress

Figure 1 Preparation, identification, and characterization of Portulaca oleracea L. -derived exosome-like nanoparticles. A: Schematic workflow for the isolation and purification of Portulaca oleracea L.-derived exosome-like nanoparticle (PELNs); B: Representative transmission electron microscopy image showing the morphology of PELNs (scale bar: 100 nm); C: Particle size distribution of PELNs as determined by nanoparticle tracking analysis; D: Total ion chromatogram (TIC) of PELNs obtained in positive ionization mode using ultra-high-performance liquid chromatography coupled with quadrupole-orbitrap high-resolution mass spectrometry (UHPLC-QE-MS); E: TIC of PELNs obtained in negative ionization mode using UHPLC-QE-MS.

Figure 2 Portulaca oleracea L. -derived exosome-like nanoparticles alleviate dextran sulfate sodium-induced colitis in mice. A: Schematic diagram of the experimental timeline; B: Body weight changes of mice throughout the experiment; C: Disease activity index scores of mice; D: Representative images and measurement of colon lengths; E: Fecal occult blood test results on day 13; F: Body weight of mice on day 13; G: Colon length of mice on day 13; H: Hematoxylin & eosin staining (× 50, × 100); I: Transmission electron microscopy observation of the ultrastructure of tight junctions and endoplasmic reticulum in intestinal epithelial cells (× 6000, × 8200); J: Representative immunofluorescence images showing Bip (red), ZO-1 (green), and DAPI (blue) staining in mouse colon tissues (× 200); K: Quantitative analysis of the relative fluorescence intensity of Bip and ZO-1 in mouse colon tissues. ^bP < 0.01, ^cP < 0.001, ^dP < 0.0001. NS: Not significant; DSS: Dextran sulfate sodium; PELNs: Portulaca oleracea L.-derived exosome-like nanoparticles; 5-ASA: 5-aminosalicylic acid.

Figure 3 Portulaca oleracea L. -derived exosome-like nanoparticles regulated inflammatory cytokine, reducing endoplasmic reticulum stress and restoring tight junctions in dextran sulfate sodium-induced colitis mice. A: The expression of interleukin (IL)-28, IL-18, IL-36, myeloperoxidase, IL-37, and IL-10 in colon tissues; B: Immunoblot bands of ZO-1, PERK, phosphorylated PERK, Bip, Occludin, ATF4, eIF2α, phosphorylated eIF2α, Claudin-4, and Claudin-2 in colon tissues; C: Statistical graphs of relative protein expression levels; D: QPCR analysis of the relative gene expression levels of PERK, eIF2α, ATF4, Bip, Claudin-2, Claudin-4, ZO-1and Occludin in colon tissues. ^aP < 0.05, ^bP < 0.01, ^cP < 0.001, ^dP < 0.0001. NS: Not significant; DSS: Dextran sulfate sodium; PELNs: Portulaca oleracea L.-derived exosome-like nanoparticles; 5-ASA: 5-aminosalicylic acid; IL: Interleukin; p-PERK: Phosphorylated PERK; p-eIF2α: Phosphorylated eIF2α.

Figure 4 Portulaca oleracea L. -derived exosome-like nanoparticles regulate the gut microbiota in dextran sulfate sodium-induced colitis mice. A: Alpha diversity of mouse gut microbiota assessed by the Ace index; B: Alpha diversity of mouse gut microbiota assessed by the Chao index; C: Principal coordinate analysis; D: Non-metric Multidimensional Scaling; E: Differential analysis of the Gut Microbiome Health Index (upper images), analysis of the microbiota dysbiosis index (lower images); F: Bar plot showing community composition at the phylum and genus levels for each group; G: Comparison of community differences at the phylum and genus levels; H: Linear discriminant analysis effect size for discriminating multi-level species differences. ^aP < 0.05, ^bP < 0.01, ^cP < 0.001 (A, B, and G: Statistical method: Kruskal-Wallis test with FDR correction; post-hoc test: Dunn's test. E: Statistical method: Wilcoxon test with multiple correction). DSS: Dextran sulfate sodium; PELNs: Portulaca oleracea L.-derived exosome-like nanoparticles; 5-ASA: 5-aminosalicylic acid; OTU: Perational taxonomic unit; PCoA: Principal coordinate analysis; LEfSe: Linear discriminant analysis effect size; NMDS: Non-metric Multidimensional Scaling.

Figure 5 Portulaca oleracea L. -derived exosome-like nanoparticles regulate gut metabolites in dextran sulfate sodium-induced colitis mice. A: Principal component analysis; B: Partial least squares-discriminant analysis; C: Volcano plot of differential metabolites; D: Bubble chart of KEGG pathway enrichment analysis for differential metabolite sets in mouse gut; E: Correlation analysis between gut microbiota and ulcerative colitis (UC)-related indicators; F: Correlation analysis between metabolites and UC-related indicators; G: Correlation analysis between gut microbiota and metabolites. ^aP < 0.05, ^bP < 0.01, ^cP < 0.001. DSS: Dextran sulfate sodium; PELNs: Portulaca oleracea L.-derived exosome-like nanoparticles; 5-ASA: 5-aminosalicylic acid; PCA: Principal component analysis; PLS-DA: Partial least squares-discriminant analysis.

Figure 6 Portulaca oleracea L. -derived exosome-like nanoparticles alleviate colitis in fecal microbiota transplantation mice. A: Schematic diagram of the experimental timeline; B: Body weight changes of mice; C: Disease activity index scores; D: Colon length; E: Fecal occult blood test results on day 24; F: Body weight on day 24; G: Colon length on day 24; H: Hematoxylin & eosin staining (× 50, × 100); I: Ultrastructure of tight junctions and endoplasmic reticulum in intestinal epithelial cells observed by transmission electron microscopy (× 6000, × 8200); J: Representative immunofluorescence images of Bip (red), ZO-1 (green), and DAPI (blue) in mouse colon tissues (× 200); K: Quantitative analysis of the relative fluorescence intensity of Bip and ZO-1 in mouse colon tissues. ^aP < 0.05, ^cP < 0.001, ^dP < 0.0001. NS: Not significant; DSS: Dextran sulfate sodium; FMT: Fecal microbiota transplantation; PELNs: Portulaca oleracea L.-derived exosome-like nanoparticles.

Figure 7 Portulaca oleracea L. -derived exosome-like nanoparticles regulated inflammatory cytokine, reducing endoplasmic reticulum stress and restoring tight junctions in fecal microbiota transplantation mice. A: The expression of interleukin (IL)-28, IL-18, IL-36, myeloperoxidase, IL-37, and IL-10 in colon tissues; B: Immunoblot bands of ZO-1, PERK, phosphorylated PERK, Bip, Occludin, ATF4, eIF2α, phosphorylated eIF2α, Claudin-4, and Claudin-2 in colon tissues; C: Statistical graphs of relative protein expression levels; D: QPCR analysis of the relative gene expression levels of ZO-1, Occludin, ATF4, Claudin-4, Bip, eIF2α, Claudin-2 and PERK in colon tissues. ^aP < 0.05, ^bP < 0.01, ^cP < 0.001, ^dP < 0.0001. DSS: Dextran sulfate sodium; FMT: Fecal microbiota transplantation; PELNs: Portulaca oleracea L.-derived exosome-like nanoparticles; IL: Interleukin; p-PERK: Phosphorylated PERK; p-eIF2α: Phosphorylated eIF2α.

Figure 8 Portulaca oleracea L. -derived exosome-like nanoparticles regulate the gut microbiota in dextran sulfate sodium-induced fecal microbiota transplantation colitis mice. A: Alpha diversity of the gut microbiota in fecal microbiota transplantation (FMT) mice assessed by the Ace index; B: Alpha diversity of the gut microbiota in FMT mice assessed by the Shannon index; C: Principal coordinate analysis; D: Non-metric Multidimensional Scaling; E: Differential analysis of the Gut Microbiome Health Index (upper images), analysis of the microbiota dysbiosis index (lower images); F: Heatmap of community composition at the genus level; G: Bar plot showing community composition at the phylum and genus levels for each group; H: Comparison of community differences at the phylum and genus levels; I: Linear discriminant analysis effect size for discriminating multi-level species differences. ^aP < 0.05, ^bP < 0.01, ^cP < 0.001 (A, B, and G: Statistical method: Kruskal-Wallis test with FDR correction; post-hoc test: Dunn's test. E: Statistical method: Wilcoxon test with multiple correction). OTU: Perational taxonomic unit; PCoA: Principal coordinate analysis; DSS: Dextran sulfate sodium; FMT: Fecal microbiota transplantation; PELNs: Portulaca oleracea L.-derived exosome-like nanoparticles; NMDS: Non-metric Multidimensional Scaling; LEfSe: Linear discriminant analysis effect size.

Figure 9 Portulaca oleracea L. -derived exosome-like nanoparticles regulate gut metabolites in dextran sulfate sodium-induced fecal microbiota transplantation colitis mice. A: Principal component analysis; B: Partial least squares-discriminant analysis; C: Volcano plot of differential metabolites; D: Bubble chart of KEGG pathway enrichment analysis for differential metabolite sets in the gut of fecal microbiota transplantation mice; E: Correlation analysis between gut microbiota and ulcerative colitis (UC)-related indicators; F: Correlation analysis between metabolites and UC-related indicators; G: Correlation analysis between gut microbiota and metabolites. The X-axis represents the enrichment ratio of differential metabolites, and the Y-axis corresponds to KEGG pathway categories. The bubble diameter is proportional to the number of differential metabolites in the pathway, and the bubble color reflects the significance level of enrichment (P value). ^aP < 0.05, ^bP < 0.01, ^cP < 0.001. DSS: Dextran sulfate sodium; FMT: Fecal microbiota transplantation; PELNs: Portulaca oleracea L.-derived exosome-like nanoparticles; PCA: Principal component analysis; PLS-DA: Partial least squares-discriminant analysis.

Figure 10  Schematic illustration: The mechanism of action for orally delivered Portulaca oleracea L. -derived exosome-like nanoparticle in the mitigation of ulcerative colitis. Portulaca oleracea L.-derived exosome-like nanoparticles exert therapeutic benefits through the modulation of the intestinal microbial community and its associated metabolites, alleviating endoplasmic reticulum stress in colitis mice, reducing inflammation, and restoring tight junction integrity in intestinal epithelial cells. ERS: Endoplasmic reticulum stress; PELNs: Portulaca oleracea L.-derived exosome-like nanoparticles.