An elemental diet protects mouse salivary glands from 5-fluorouracil-induced atrophy

Abstract

An elemental diet (ED) reduces adverse effects of chemotherapy, including oral mucositis, in patients with cancer. However, the detailed mechanism(s) of the healing effects of an ED remains unclear. In the present study, the protective effects of the ED, Elental^®, were examined against 5-fluorouracil (5-FU)-induced oral mucositis and salivary gland atrophy in mice. Mucositis was induced in female ICR mice by injection of 5-FU. The mice were orally administered Elental^® (ED group) or saline (control group). After treatment, the mice body weight, salivary gland weight and the histological changes in the salivary gland granular duct area were monitored. The mice body weight remained stable in the ED group, but was significantly decreased in the control group. Moreover, the salivary gland weight was higher in the ED group compared with the control group. In addition, the salivary gland granular duct area cells were larger in the ED group compared with the control group. Whole transcriptome analysis and network analysis were conducted to understand the mechanisms of action of Elental^® against oral mucositis. Whole transcriptome analysis and Ingenuity Pathways Analysis data suggested that Elental^® contributed to the recovery of mitochondrial function in 5-FU-damaged salivary glands. Immunohistochemical analysis of salivary gland tissue demonstrated that the expression of cytochrome c oxidase subunit 4 and epidermal growth factor were higher in the ED group compared with the control group. Next, the rate of apoptosis in the salivary glands was examined using terminal deoxynucleotidyl transferase-mediated nick end labeling (TUNEL) assays. The number of TUNEL-positive cells in the salivary glands was lower in the ED group compared with the control group. These findings suggested that Elental^® may protect mouse salivary glands from 5-FU-induced atrophic changes, which suggests that ED treatment may improve xerostomia and alleviate oral mucositis in patients with cancer receiving 5-FU-based chemotherapy.

Keywords: 5-fluorouracil; elemental diet; granular duct; network analysis; salivary gland; whole transcriptome analysis.

Figure 1.

Effect of an ED on body weight of mice injected with 5-FU. Saline-treated (control) mice and Elental^®-treated (ED) mice were injected with 5-FU. The body weight of ED-treated mice remained stable during the treatment period. The body weight of the control group mice gradually decreased over time, and a significant difference was observed on day 21 between the ED group and the control group. n=3 mice per group. *P<0.01 vs. control group. ED, elemental diet; 5-FU, 5-fluorouracil.

Figure 2.

Effects of ED on salivary glands of mice injected with 5-FU. Saline-treated (control) mice and Elental^®-treated (ED) mice were injected with 5-FU. (A) Salivary gland weight was measured and was significantly higher in the ED group compared with the control group. (B) Histological changes in salivary glands of mice observed by HE staining. In control tissue, salivary acini cells (yellow line) and granular duct cells (arrow) atrophied upon treatment with 5-FU (magnification, ×200; scale bar, 50 µm). By contrast, mice in the ED group appeared to recover from 5-FU-induced atrophy. n=3 mice per group. *P<0.01 vs. control group. ED, elemental diet; 5-FU, 5-fluorouracil; HE, hematoxylin and eosin.

Figure 3.

Data obtained from whole transcriptome analysis, PCA and IPA of 5-FU-injected ED) mice and saline-treated (control) mice. On day 21 of treatment, the mice were sacrificed and the salivary glands were collected for whole transcriptome analysis to identify the differentially expressed genes in the salivary glands from the ED group compared with the control group. (A) PCA was used to verify the data quality. PC1 and PC2 were plotted on the x-axis and y-axis, respectively, to draw the scatter diagram, where each point represents a sample. E1, E2 and E3 represent the ED group; and C1, C2 and C3 represent the control group. (B) Left panel: A volcano plot showing each differentially expressed gene in the ED group compared with the control group. The significantly upregulated genes are shown in red, and the significantly downregulated genes are shown in green. Right panel: IPA analysis of the upregulated (>1.5-fold) genes identified with the volcano plot indicated that these genes were mostly associated with oxidative phosphorylation and mitochondrial dysfunction pathways. (C) IPA analysis of the differentially expressed genes in the salivary glands from the ED group compared with the control group, which were associated with the oxidative phosphorylation pathway-related network. (D) Mitochondrial function-related network constructed from the IPA data of the differentially expressed genes in the salivary glands from the ED group compared with the control group (large block letters refer to the upregulated genes). PCA, principal component analysis; IPA, Ingenuity Pathways Analysis; ED, elemental diet; 5-FU, 5-fluorouracil; PC, principal component; E, ED group; C, control group.

Figure 4.

Effect of an ED on the expression of COX IV and EGF in salivary glands of ED- or saline-treated (control) mice injected with 5-FU as observed by immunohistochemical staining. (A) Expression of COX IV was higher in the salivary glands from ED mice compared with control mice. (B) EGF expression was higher in the salivary glands from ED mice compared with the control mice. Magnification, ×200. Scale bar, 50 µm. ED, elemental diet; COX IV, COX subunit 4; EGF, epidermal growth factor.

Figure 5.

Effect of an ED on salivary gland apoptosis in ED- or saline-treated (control) mice injected with 5-FU. (A) A TUNEL assay was performed to detect apoptotic cells in control and ED-treated salivary glands. Arrows indicate apoptotic cells (magnification, ×100; scale bars, 100 µm. (B) TUNEL-positive cells in the salivary glands of the control group mice were significantly higher compared with those from the ED group mice. n=3 mice per group. ^#P<0.01 vs. control group. ED, elemental diet; 5-FU, 5-fluorouracil.