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. 2012;7(3):e31808.
doi: 10.1371/journal.pone.0031808. Epub 2012 Mar 7.

5-Fluorouracil induced intestinal mucositis via nuclear factor-κB activation by transcriptomic analysis and in vivo bioluminescence imaging

Chung-Ta Chang  1 , Tin-Yun Ho, Ho Lin, Ji-An Liang, Hui-Chi Huang, Chia-Cheng Li, Hsin-Yi Lo, Shih-Lu Wu, Yi-Fang Huang, Chien-Yun Hsiang
Affiliations

5-Fluorouracil induced intestinal mucositis via nuclear factor-κB activation by transcriptomic analysis and in vivo bioluminescence imaging

Chung-Ta Chang et al. PLoS One. 2012.

Abstract

5-Fluorouracil (5-FU) is a commonly used drug for the treatment of malignant cancers. However, approximately 80% of patients undergoing 5-FU treatment suffer from gastrointestinal mucositis. The aim of this report was to identify the drug target for the 5-FU-induced intestinal mucositis. 5-FU-induced intestinal mucositis was established by intraperitoneally administering mice with 100 mg/kg 5-FU. Network analysis of gene expression profile and bioluminescent imaging were applied to identify the critical molecule associated with 5-FU-induced mucositis. Our data showed that 5-FU induced inflammation in the small intestine, characterized by the increased intestinal wall thickness and crypt length, the decreased villus height, and the increased myeloperoxidase activity in tissues and proinflammatory cytokine production in sera. Network analysis of 5-FU-affected genes by transcriptomic tool showed that the expression of genes was regulated by nuclear factor-κB (NF-κB), and NF-κB was the central molecule in the 5-FU-regulated biological network. NF-κB activity was activated by 5-FU in the intestine, which was judged by in vivo bioluminescence imaging and immunohistochemical staining. However, 5-aminosalicylic acid (5-ASA) inhibited 5-FU-induced NF-κB activation and proinflammatory cytokine production. Moreover, 5-FU-induced histological changes were improved by 5-ASA. In conclusion, our findings suggested that NF-κB was the critical molecule associated with the pathogenesis of 5-FU-induced mucositis, and inhibition of NF-κB activity ameliorated the mucosal damage caused by 5-FU.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. Histological examination of the small intestine following 5-FU administration.
BALB/c mice were intraperitoneally administered with PBS (mock) or 5-FU and sacrificed 2 days later. (A) Microscopic features of the jejunum. Sections were stained with H&E and observed using light microscopy. Magnification 100×. Photos are representative images. (B) Intestinal morphometry of intestinal wall thickness, crypt length, and villus height. Six mice in each group were sacrificed for the morphometry analysis. Three intestinal walls, crypts, and villi in 3 longitudinal sections per mouse were counted. Results are expressed as relative length, which is presented as a comparison with the length or thickness relative to mock. Values are mean ± standard error. **p<0.01, ***p<0.001, compared with mock.
Figure 2
Figure 2. Network analysis of 5-FU-affected genes in the small intestine.
Upregulated genes are marked with red circles/disks. Downregulated genes are marked with blue circles/disks. Cyan lines indicate the fragments of canonical pathways.
Figure 3
Figure 3. NF-κB-dependent bioluminescence in living mice and individual organs following 5-FU administration.
(A) In vivo imaging. Transgenic mice were injected intraperitoneally with PBS or 5-FU and imaged at indicated periods. Results are expressed as relative intensity, which is presented as the comparison with the NF-κB-dependent bioluminescent signal relative to mock. Values are mean ± standard error (n = 6 per group). *p<0.05, compared with mock. (B) Ex vivo imaging. Transgenic mice were injected intraperitoneally with PBS (mock) or 5-FU. Two days later, mice were sacrificed and organs were subjected to image. The color overlay on the image represents the photon/sec emitted from the organs, as indicated by the color scale. Photos are representative images (n = 6 per group). (C) Quantification of photon emission from the organs. Values are mean ± standard error (n = 6 per group). **p<0.01, compared with mock. (D) NF-κB-dependent bioluminescence in the intestine following 5-FU and/or 5-ASA administration. Transgenic mice were administered with 5-FU and/or 5-ASA and imaged 2 days later. The color overlay on the image represents the photon/sec emitted from the intestine, as indicated by the color scale. Photos are representative images (n = 6 per group). Quantification of photon emission from the intestine was shown on the top. Values are mean ± standard error. *p<0.05, compared with mock. # p<0.05, compared with 5-FU treatment.
Figure 4
Figure 4. Immunohistochemical staining and MPO activity of jejunum and cytokine ELISA of sera following 5-FU and/or 5-ASA administration.
Transgenic mice were administered with 5-FU and/or 5-ASA and sacrificed 2 days later. (A) Sections were stained by immunohistochemistry using antibody against NF-κB, IL-1β, or TNF-α. Magnification 100×. Photos are representative images (n = 6 per group). (B) MPO activity assay. Frozen jejunum was homogenized and MPO activity in the tissue was analyzed. Values are mean ± standard error. *p<0.05, compared with mock. (C) Cytokine ELISA. The levels of IL-1β and TNF-α were analyzed by cytokine ELISA. Values are mean ± standard error. *p<0.05, ***p<0.001, compared with mock. ### p<0.001, compared with 5-FU treatment.
Figure 5
Figure 5. Histological examination of the small intestine following 5-FU and/or 5-ASA administration.
Transgenic mice were administered with 5-FU and/or 5-ASA and sacrificed 2 days later. (A) Microscopic features of the jejunum. Sections were stained with H&E and observed using light microscopy. Magnification 40×. Photos are representative images (n = 6 per group). (B) Intestinal morphometry of intestinal wall thickness, crypt length, and villus height. Six mice in each group were sacrificed for the morphometry analysis. Three intestinal walls, crypts, and villi in 3 longitudinal sections per mouse were counted. Results are expressed as relative length, which is presented as a comparison with the length or thickness relative to mock. Values are mean ± standard error. **p<0.01, compared with mock. ## p<0.01, compared to 5-FU treatment.
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