Modulation of gut microbiota and immune response by soy peptides mitigates irinotecan induced intestinal toxicity

Abstract

Introduction: Irinotecan (CPT-11), a cornerstone chemotherapeutic agent for colorectal and pancreatic cancers, is limited by severe gastrointestinal toxicities, particularly diarrhea, which compromises treatment adherence and patient quality of life. Soy peptides (SPs), bioactive compounds with anti-inflammatory and prebiotic properties, have shown potential in enhancing intestinal barrier function. This study investigates SPs' protective effects against irinotecan-induced intestinal injury, focusing on microbiota modulation, immune regulation, and mucosal repair.

Methods: Female C57BL/6 mice were randomly divided into four groups (n = 10/group): Control, Irinotecan, Pre-SPs+ Irinotecan, and SPs+ Irinotecan. Diarrhea severity and body weight changes were monitored daily. Small intestinal injury was evaluated by hematoxylin and eosin (H&E) staining with epithelial damage scoring, while intestinal barrier integrity was assessed via Western blotting (WB) and immunohistochemistry. Serum levels of inflammatory cytokines (TNF-α and IL-6) were quantified using ELISA, and neutrophil infiltration was measured by flow cytometry. Fecal samples were subjected to 16S rRNA sequencing to analyze gut microbiota composition.

Results: SPs intervention significantly reduced the incidence of diarrhea (P < 0.05) and attenuated body weight loss (P < 0.05) in mice. Histological analysis demonstrated that SPs restored intestinal architecture, as evidenced by reduced epithelial damage scores (P < 0.05), increased expression of tight junction proteins (occludin and ZO-1), and improved intestinal permeability (P < 0.05). Gut microbiota profiling revealed that irinotecan-induced dysbiosis was characterized by decreased α-diversity and enrichment of pathogenic taxa. SPs treatment restored microbial diversity and significantly elevated the abundance of beneficial genera, including Lactobacillus and Bifidobacterium (P < 0.05). Immunological assays further indicated that SPs suppressed pro-inflammatory cytokine levels (TNF-α and IL-6, P < 0.001) and reduced neutrophil infiltration (P < 0.05).

Discussion: These findings suggest that soy peptides protect against irinotecan-induced intestinal toxicity through multiple mechanisms, including microbiota regulation, immune modulation, and intestinal barrier restoration, highlighting their potential as a therapeutic candidate for chemotherapy-induced intestinal damage.

Keywords: diarrhea; gut microbiota; immune modulation; intestinal damage; irinotecan; soy peptides.

FIGURE 1

Soybean Peptides Alleviate Weight Loss and Diarrhea Induced by CPT-11 in Mice (A) The process of animal modeling. (B) Diarrhea Score in Mice. (C) Body Weight Change in Mice. Data represent mean ± standard deviation (SD). n = 10 per group. *P < 0.05, **P < 0.01.

FIGURE 2

Soybean peptides protect mouse intestinal tissues from irinotecan-induced damage. (A) H&E staining of the ileal tissue in mice (30×). (B) Damage score of the ileal mucosal tissue. (C) Villus/crypt ratio. (D) Inflammatory infiltration. (E) Epithelial inflammation. (F) Expression of IL-6 in intestinal tissue. (G) Expression of TNF-α in intestinal tissue. Each group, n = 10. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

FIGURE 3

Soybean Peptides Maintain Intestinal Permeability in Mice. (A) Western blot analysis shows the expression of ZO-1 and Occludin in mouse ileum tissue. (B,C) ImageJ analysis indicates that the expression of ZO-1 and Occludin in the soybean peptide group was significantly higher than that in the irinotecan group, approaching the levels of the control group. Data are presented as mean ± SD, n ≥ 3 per group. (D,E) Representative immunohistochemical images of intestinal tissue show the expression of Occludin and ZO-1, with staining intensity quantified for evaluation (scale bar = 50 μm; magnification: ×25). *P < 0.05, **P < 0.01, ***P < 0.001.

FIGURE 4

Soybean Peptides Restore Irinotecan-Induced Dysbiosis of the Gut Microbiota. (A) The α-diversity of the gut microbiota in the four groups of mice was evaluated using the Chao1, Shannon, and Simpson indices. (B) Principal coordinate analysis (PCoA), based on the Bray-Curtis distance matrix, shows differences in the gut microbiota structure among the different treatment groups. (C) The bar chart displays the differences in the gut microbiota composition at the phylum level. (D) The bar chart shows the differences in the gut microbiota composition at the genus level.

FIGURE 5

Differential Microbial Taxa and Metabolic Pathways Between Groups. (A,B) Differential bacterial taxa identified by LEfSe analysis: (A) LDA bar chart (LDA ≥3) showing significantly different bacterial taxa. (B) Taxonomic cladogram, where the node size corresponds to the average relative abundance of taxonomic units. Hollow nodes indicate no significant differences between groups, while colored nodes represent significant differences, with higher abundance in the respective group. (C–E) Differential metabolic pathway analysis between groups: (C) Differential metabolic pathways between the Control and Irinotecan groups. (D) Differential metabolic pathways between the Irinotecan and Pre-SPs + Irinotecan groups. (E) Differential metabolic pathways between the Irinotecan and SPs + Irinotecan groups. Note: Orange (P < 0.001), Yellow (P < 0.01), Blue (P < 0.05). Positive values on the x-axis indicate upregulation relative to the control group, while negative values indicate downregulation.

FIGURE 6

Soybean peptides reduce inflammation in mice. (A,B) Soybean peptides restore the reduction in neutrophil count induced by CPT-11 treatment. Note: *P < 0.05, **P < 0.01.

FIGURE 7

Soybean Peptides Alleviate Irinotecan (CPT-11)-Induced Hepatotoxicity. (A) Expression levels of AST in serum. (B) Expression levels of ALT in serum. (C) Expression levels of ALP in serum. (D) Expression levels of TBIL in serum. Note: Each group n = 10. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.