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, 9 (1), 19533

Analysis of Free Radical Production Capacity in Mouse Faeces and Its Possible Application in Evaluating the Intestinal Environment: A Pilot Study

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Analysis of Free Radical Production Capacity in Mouse Faeces and Its Possible Application in Evaluating the Intestinal Environment: A Pilot Study

Yoshihisa Wakita et al. Sci Rep.

Abstract

Complex interplay between the intestinal environment and the host has attracted considerable attention and has been well studied with respect to the gut microbiome and metabolome. Oxygen free radicals such as superoxide and the hydroxyl radical (OH) are generated during normal cellular metabolism. They are toxic to both eukaryotic and prokaryotic cells and might thus affect intestinal homeostasis. However, the effect of oxygen free radicals on the intestinal environment has not been widely studied. Herein, we applied electron spin resonance spectroscopy with spin trapping reagents to evaluate oxygen free radical production capacity in the intestinal lumen and the faeces of mice. OH was generated in faeces and lumens of the small and large intestines. There were no remarkable differences in OH levels between faeces and the large intestine, suggesting that faeces can be used as alternative samples to estimate the OH production capacity in the colonic contents. We then compared free radical levels in faecal samples among five different mouse strains (ddY, ICR, C57BL/6, C3H/HeJ, and BALB/c) and found that strain ddY had considerably higher levels than the other four strains. In addition, strain ddY was more susceptible to dextran sulphate sodium-induced colitis. These differences were possibly related to the relative abundance of the gut bacterial group Candidatus Arthromitus, which is known to modulate the host immune response. From these results, we suggest that the production capacity of oxygen free radicals in mouse faeces is associated with intestinal homeostasis.

Conflict of interest statement

This research did not receive specific grant from any funding agency in the public, commercial, or not-for-profit sectors. Y.W., A.S., H. Kaneda, S.S. and Y.T. are employees of SAPPORO HOLDINGS LTD. The remaining authors have no conflict of interest to disclose.

Figures

Figure 1
Figure 1
Oxygen free radical production capacity in faeces and the small and large intestines. (a) Schematic diagram of ligated intestinal loop assay. ddY mice were anesthetized with isoflurane gas. After opening the abdomen, both ends of the large or small intestine were bound with surgical thread, and POBN solution was injected. The contents were collected and centrifuged, and the supernatants were analysed by electron spin resonance (ESR). Faeces were reacted with POBN solution and analysed similarly. (b) ESR spectra from faeces and the small and large intestines. The center line (from the bottom to the top of the peak) marked with an asterisk (*) was used to calculate the relative amounts of OH. (c) Relative amounts of OH. Data are shown as mean values ± SD. N = 6–7 per group. One-way ANOVA showed no significant differences among OH levels in the faeces, small intestine, and large intestine. (d) A typical ESR spectrum from faeces obtained with the spin-trapping agent CYPMPO. The peak-to-peak intensities of the line marked with an asterisk (*) and double asterisk (**) were used to calculate relative amounts of OH and O2•−, respectively. (e) Relative amounts of OH in different mouse strains. Data are shown as mean values ± SD (n = 6 per group). There were significant differences for ddY vs. ICR, C57BL/6, C3H/HeJ, and BALB/c. *p < 0.001 (vs. ddY). (f) Relative levels of OH in germ free (GF) and ex-germ-free (Ex-GF) mice Ex-GF mice were inoculated with faeces obtained from SPF C57BL/6 mice. Data are shown as mean values ± SD (n = 10 per group). *p < 0.001.
Figure 2
Figure 2
Structure of intestinal microbiome in different mouse strains. Amplicon sequencing was performed using the V1/V2 regions of the 16S rRNA gene, followed by analysis using the QIIME pipeline. (a) Unweighted unifrac analysis of intestinal microbiome. (b) Weighted unifrac analysis of intestinal microbiome. (c) Relative abundance of intestinal microbiota at the genus level. Genera for which the abundance was greater than 2% are shown.
Figure 3
Figure 3
Correlation between relative abundance of genera and relative peak height of OH. Correlation between relative abundance of 24 genera (Fig. S1) and relative peak heights of OH were evaluated by Spearman’s rank correlation coefficient. A probability value of less than 0.0021 (0.05/24) was considered statistically significant. The genera for which the probability value was less than 0.0021 are shown.
Figure 4
Figure 4
A comparison of responses to dextran sulphate sodium (DSS)-induced colitis among five different mouse strains. (a) Change in body weight on days 4 and 7. (b) Cumulative survival rate. Seven-week-old mice were used (n = 10 per group). Mice were allowed free access to 4% DSS water.

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