Radiation therapy induced intestinal barrier damage and repair process - differences in salivary metabolites and monitoring of intestinal barrier function

Abstract

Purpose: Colorectal cancer (CRC) is still one of the most common malignant tumors, with gradual increase in its annual morbidity and mortality. But most cases are diagnosed in the late stage. For stage II-III cancer, clinical guidelines recommend surgery following neoadjuvant radiation therapy at ≥6 weeks after the last radiotherapy is completed. However, radiotherapy may impair intestinal mucosal barrier function, especially the biological and immune barriers, accompanied by perioperative complications. This study was conducted to investigate the changes, repair patterns, and potential mechanisms in patients after radiotherapy.

Methods: This study detected inflammatory factors in postoperative intestinal mucosal tissue and serum, as well as metabolites in saliva samples, and collected hematoxylin-eosin (HE)-stained pathological images in CRC patients who had received and did not receive radiotherapy.

Results: The results showed that after radiotherapy, there were significantly impaired intestinal mucosal tissue structure; obviously elevated inflammatory factors in intestinal mucosal tissue and blood; as well as upregulation/downregulation of metabolites in saliva samples.

Conclusion: In conclusion, findings in this study may provide potential reference for predicting the recovery of intestinal mucosa and selecting the optimal timing for surgery after radiotherapy. In addition, this study will benefit the understanding and reduction of perioperative complications caused by intestinal barrier damage.

Keywords: biological barrier; damage and repair; immune barrier; intestinal mucosal barrier; radiotherapy; rectal cancer; salivary metabolites.

Figure 1

The above are the more representative pictures of HE stained slices of normal tissue intestinal mucosa distal to the tumor in the postoperative period of patients with rectal cancer who underwent surgery in our center, (A-D) are 60 days ± 3 days, 75 days ± 3 days, and 90 days ± 3 days after no radiotherapy versus radiotherapy, respectively.

Figure 2

The graph shows the cytokine levels in the intestinal mucosal tissues of the four groups of samples. Panel (A) indicates INF-γ, Panel (B) indicates TGF-β, X-axis groups, where group 1 (no radiotherapy group), group 2 (60 ± 3 days after radiotherapy), group 3 (75 ± 3 days after radiotherapy), and group 4 (90 ± 3 days after radiotherapy). y-axis indicates the total grayscale values of the WB detection bands for each cytokine. Where * denotes p< 0.05; ** denotes p < 0.01; Δ denotes p > 0.05. Panel (C) shows the electrophoretic images of cytokines detected by protein blotting, with different cytokines indicated on the right side and different groups labeled on the top.

Figure 3

The upper graph demonstrates the cytokine content in patients’ serum, where (A-D) denote the four inflammatory factors, IL-1β, IL-6, IL-17, and INF-γ, respectively, with vertical coordinates denoting the content, and horizontal coordinates denoting the groups, Group 1 (8–12 weeks after radiotherapy) and Group 2 (no radiotherapy group). Where * indicates p< 0.05; ** indicates p < 0.01; Δ indicates p > 0.05.

Figure 4

Panel (A) is the grouped principal component analysis, PC1 represents the first principal component, PC2 represents the second principal component, and the percentage represents the variance explained by the principal component to the data set; each point in the figure represents a sample, and samples in the same group are represented by the same color, and Group is the subgroup. Panel (B) is the differential metabolite clustering heatmap, the horizontal is the information of the samples, the vertical is the information of the differential metabolites, and Group is the subgroup. Group is the grouping, and different colors are the colors filled with different values obtained from the normalization of different relative contents (red for high content, green for low content). Panel (C) is the differential metabolite volcano plot, each point represents a metabolite, where green points represent down-regulated differential metabolites and red points represent up-regulated differential metabolites; the horizontal coordinate represents the relative content of a metabolite in the two groups of samples Under VIP + FC + P-value screening condition: the vertical coordinate represents the significance level of difference (-log1010P-value), and the size of the dots represents the VIP value. Panel (D) is the bar chart of differential metabolites, and the horizontal coordinate is the log22FC of the differential metabolites, i.e., the value of the multiplicity of difference of differential metabolites logarithmically based on the base of 2, and the vertical coordinate is the differential metabolites. The vertical coordinate is the differential metabolite. Red color represents up-regulation of metabolite content and green color represents down-regulation of metabolite content.