Panaxynol improves crypt and mucosal architecture, suppresses colitis-enriched microbes, and alters the immune response to mitigate colitis

Abstract

Ulcerative colitis (UC) is an idiopathic inflammatory disease of the large intestine, which impacts millions worldwide. Current interventions aimed at treating UC symptoms can have off-target effects, invoking the need for alternatives that may provide similar benefits with less unintended consequences. This study builds on our initial data, which showed that panaxynol-a novel, potent, bioavailable compound found in American ginseng-can suppress disease severity in murine colitis. Here we explore the underlying mechanisms by which panaxynol improves both chronic and acute murine colitis. Fourteen-week-old C57BL/6 female mice were either given three rounds of dextran sulfate sodium (DSS) in drinking water to induce chronic colitis or one round to induce acute colitis. Vehicle or panaxynol (2.5 mg/kg) was administered via oral gavage three times per week for the study duration. Consistent with our previous findings, panaxynol significantly (P < 0.05) improved the disease activity index and endoscopic scores in both models. Using the acute model to examine potential mechanisms, we show that panaxynol significantly (P < 0.05) reduced DSS-induced crypt distortion, goblet cell loss, and mucus loss in the colon. 16S Sequencing revealed panaxynol altered microbial composition to suppress colitis-enriched genera (i.e., Enterococcus, Eubacterium, and Ruminococcus). In addition, panaxynol significantly (P < 0.05) suppressed macrophages and induced regulatory T-cells in the colonic lamina propria. The beneficial effects of panaxynol on mucosal and crypt architecture, combined with its microbial and immune-mediated effects, provide insight into the mechanisms by which panaxynol suppresses murine colitis. Overall, this data is promising for the use of panaxynol to improve colitis in the clinic.NEW & NOTEWORTHY In the current study, we report that panaxynol ameliorates chemically induced murine colitis by improving colonic crypt and mucosal architecture, suppressing colitis-enriched microbes, reducing macrophages, and promoting the differentiation of regulatory T-cells in the colonic lamina propria. This study suggests that this novel natural compound may serve as a safe and effective treatment option for colitis patients.

Keywords: American ginseng; goblet cells; macrophages; microbiota; regulatory T-cells.

Graphical abstract

Figure 1.

Panaxynol improves disease severity and symptomology in chronic murine colitis. A: schematic demonstrating experimental design, disease induction, and treatment administration for the chronic colitis model. Symptoms quantified using disease activity index (n = 6 Con; 19 DSS + Veh; 19 DSS + Pax): body weight (g) (B), stool consistency score (C), rectal bleeding score (D), and overall symptom score (E). F: representative images from end-of-study endoscopies. G: endoscopic scores (n = 2 or 3/group). One-way ANOVA. Con, control; DSS, dextran sulfate sodium; Pax, panaxynol; Veh, vehicle. †Significant (P < 0.05) difference Control vs. DSS + Veh; ǂsignificant (P < 0.05) difference Control vs. DSS + Pax; *significant (P < 0.05) difference DSS + Veh vs. DSS + Pax. n, animals per group.

Figure 2.

Panaxynol improves disease severity and symptomology in acute murine colitis. A: schematic demonstrating experimental design, disease induction, and treatment administration for the acute colitis model. Symptoms quantified using disease activity index (n = 10 Con; 16 DSS + Veh; 16 DSS + Pax): body weight (g) (B), stool consistency score (C), rectal bleeding score (D), and overall symptom score (E). F: representative images from end-of-study endoscopies. G: endoscopic scores (n = 2 or 3/group). One-way ANOVA. Con, control; DSS, dextran sulfate sodium; Pax, panaxynol; Veh, vehicle. †Significant (P < 0.05) difference Control vs. DSS + Veh; ǂsignificant (P < 0.05) difference Control vs. DSS + Pax; *significant (P < 0.05) difference DSS + Veh vs. DSS + Pax. n, animals per group.

Figure 3.

Panaxynol reduces colonic crypt distortion due to DSS and improves mucosal architecture. A: representative images of hematoxylin and eosin (H&E) staining of the distal colon taken at ×20 (scale bar = 50 µm). B: representative transmission electron microscopy (TEM) images of the distal colon (scale bar = 2 µm), showing intercellular edema (yellow arrows). Average colonic crypt width (C) and crypt width distribution curve (D) (n = 2 Con; 4 DSS + Veh; 4 DSS + Pax). E: representative scanning electron microscopy (SEM) images of the distal colon taken at ×700 (scale bar = 20 µm), showing mucosal flattening (red arrows). F: representative SEM images of the distal colon taken at ×3,000 (scale bar = 5 µm), showing crypts (white arrows). One-way ANOVA. Con, control; DSS, dextran sulfate sodium; Pax, panaxynol; Veh, vehicle. †Significant (P < 0.05) difference Control vs. DSS + Veh; ǂsignificant (P < 0.05) difference Control vs. DSS + Pax; *significant (P < 0.05) difference DSS + Veh vs. DSS + Pax. n, animals per group.

Figure 4.

Panaxynol inhibits the loss of goblet cells per crypt and preserves mucus loss due to DSS. A: representative images of Alcian Blue (AB) staining of the distal colon taken at ×40 (scale bar = 25 µm). B: average goblet cell count per crypt (n = 2 Con; 4 DSS + Veh; 4 DSS + Pax). C: representative scanning electron microscopy (SEM) images of the distal colon taken at ×2,000 (scale bar = 10 µm), showing mucus (orange arrows). D: representative SEM images of the distal colon taken at ×6,000 (scale bar = 2 µm), showing goblet cells (purple arrows). One-way ANOVA. Con, control; DSS, dextran sulfate sodium; Pax, panaxynol; Veh, vehicle. †Significant (P < 0.05) difference Control vs. DSS + Veh; *significant (P < 0.05) difference DSS + Veh vs. DSS + Pax. n, animals per group.

Figure 5.

Panaxynol alters microbial beta diversity and taxonomic structure. Results from 16S sequencing (n = 5 Con; 4 DSS + Veh; 5 DSS + Pax). A: microbial α-diversity assessed by Shannon index. B: microbial α-diversity assessed by Simpson index. C: microbial β-diversity assessed by PCoA and PERMANOVA (P value = 0.001). D: bar plot for phylum level taxonomy. E: bar plot for family level taxonomy. F: bar plot for genus level taxonomy. G and H: heat trees showing relative enrichment across taxonomy between groups, whereby red signifies increased relative abundance and blue signifies decreased relative abundance: Comparison between Control and DSS + Veh (G); comparison between DSS + Veh and DSS + Pax (H). Con, control; DSS, dextran sulfate sodium; Pax, panaxynol; Veh, vehicle. n, animals per group.

Figure 6.

Panaxynol suppresses colitis-enriched microbes. Results from 16S sequencing (n = 5 Con; 4 DSS + Veh; 5 DSS + Pax). A: heat map showing changes in microbial community composition at the genus level, whereby the top 17 taxa are displayed. B: LEFSe was used to detect relative enrichment of genera in each treatment group. C–L: individual genera that were significantly altered are shown. One-way ANOVA. Con, control; DSS, dextran sulfate sodium; Pax, panaxynol; Veh, vehicle. †Significant (P < 0.05) difference Control vs. DSS + Veh; ǂsignificant (P < 0.05) difference Control vs. DSS + Pax; *significant (P < 0.05) difference DSS + Veh vs. DSS + Pax. n, animals per group.

Figure 7.

Panaxynol suppresses macrophages in the colonic lamina propria. A: representative flow plots of gating strategy for macrophages: colonic lamina propria (LP) cells were gated for nondebris singlets and considered live immune cells with ZombieGreenNeg/Low and CD45⁺. From the live CD45⁺ population, CD11b⁺CD68⁺ cells were identified as overall macrophages. From the CD11b⁺CD68⁺ macrophage population, macrophage phenotypes were determined based on CD206 and CD11c expression—CD206⁻CD11c⁺ as M1 macrophages and CD206⁺CD11c⁻ cells as M2 macrophages. Macrophage phenotypes in the colonic LP (n = 3 Con; 4 DSS + Veh; 4 DSS + Pax): % of live CD45⁺ immune cells (B), total counts of live CD45⁺ immune cells (C), % of CD11b⁺CD68⁺ overall macrophages (D), total counts of CD11b⁺CD68⁺ overall macrophages (E), % of CD206⁻CD11c⁺ M1 macrophages (F), total counts of CD206⁻CD11c⁺ M1 macrophages (G), % of CD206⁺CD11c⁻ M2 macrophages (H), and total counts of CD206⁺CD11c⁻ M2 macrophages (I). J: representative F4/80 immunofluorescence images of the distal colon taken at ×20 (scale bar = 50 µm). K: representative transmission electron microscopy (TEM) images, showing phagocytic immune cells in the distal colon (scale bar = 1 µm and 2 µm). One-way ANOVA. Con, control; DSS, dextran sulfate sodium; Pax, panaxynol; Veh, vehicle. †Significant (P < 0.05) difference Control vs. DSS + Veh; ǂsignificant (P < 0.05) difference control vs. DSS + Pax; *significant (P < 0.05) difference DSS + Veh vs. DSS + Pax. n, animals per group.

Figure 8.

Panaxynol increases regulatory T-cells in the colonic lamina propria. A: representative flow plots of gating strategy for T-cells: colonic lamina propria (LP) cells were gated for nondebris singlets and considered immune cells with CD45⁺. From the CD45⁺ immune cell population, CD4⁺ cells were identified as helper T-cells. From the CD45⁺CD4⁺ population, TCRβ⁺FoxP3⁺ cells were identified as regulatory T-cells. T-cell phenotypes in the colonic LP (n = 5 Con; 5 DSS + Veh; 6 DSS + Pax): relative percentage of CD45⁺CD4⁺ helper T-cells in the colonic LP (B) and relative percentage of TCRβ⁺FoxP3⁺ regulatory T-cells in the colonic LP (C). One-way ANOVA. Con, control; DSS, dextran sulfate sodium; Pax, panaxynol; Veh, vehicle. †Significant (P < 0.05) difference Control vs. DSS + Veh; ǂsignificant (P < 0.05) difference Control vs. DSS + Pax; *significant (P < 0.05) difference DSS + Veh vs. DSS + Pax. n, animals per group.