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. 2022 Dec 22:13:1051200.
doi: 10.3389/fmicb.2022.1051200. eCollection 2022.

Characteristics of intestinal microbiota in C57BL/6 mice with non-alcoholic fatty liver induced by high-fat diet

Guangwen Yan  1 , Shuaibing Li  2   3 , Yuhang Wen  2   3 , Yadan Luo  2   3 , Jingrong Huang  2   3 , Baoting Chen  2   3 , Shuya Lv  2   3 , Lang Chen  2   3 , Lvqin He  2   3 , Manli He  2   3 , Qian Yang  2   3 , Zehui Yu  2   3 , Wudian Xiao  2   3 , Yong Tang  3   4 , Weiyao Li  5 , Jianhong Han  2   3 , Fangfang Zhao  5 , Shumin Yu  5 , Fang Kong  2   3 , Benazir Abbasi  6 , Hongmei Yin  1 , Congwei Gu  2   3   5
Affiliations

Characteristics of intestinal microbiota in C57BL/6 mice with non-alcoholic fatty liver induced by high-fat diet

Guangwen Yan et al. Front Microbiol. .

Abstract

Introduction: As a representation of the gut microbiota, fecal and cecal samples are most often used in human and animal studies, including in non-alcoholic fatty liver disease (NAFLD) research. However, due to the regional structure and function of intestinal microbiota, whether it is representative to use cecal or fecal contents to study intestinal microbiota in the study of NAFLD remains to be shown.

Methods: The NAFLD mouse model was established by high-fat diet induction, and the contents of the jejunum, ileum, cecum, and colon (formed fecal balls) were collected for 16S rRNA gene analysis.

Results: Compared with normal mice, the diversity and the relative abundance of major bacteria and functional genes of the ileum, cecum and colon were significantly changed, but not in the jejunum. In NAFLD mice, the variation characteristics of microbiota in the cecum and colon (feces) were similar. However, the variation characteristics of intestinal microbiota in the ileum and large intestine segments (cecum and colon) were quite different.

Discussion: Therefore, the study results of cecal and colonic (fecal) microbiota cannot completely represent the results of jejunal and ileal microbiota.

Keywords: 16S rDNA sequencing; high-fat diet; intestinal microbiota; mice; non-alcoholic fatty liver disease.

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Conflict of interest statement

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Figures

Figure 1
Figure 1
The body weight and Histopathology with Control and NAFLD mice. (A) Body weight changes in ND or HFD fed mice over 16 weeks. (B) Representative pictures of hematoxylin and eosin (H&E) staining for liver (100× and 400×), the green arrows indicate hepatic cell steatosis, and the yellow arrows indicate hepatic cell balloon-like changes. (C,D) Representative pictures of Oil red O staining for liver (400×) and quantitative analysis of lipid droplets relative amount. (E,F) Representative pictures of Masson staining for liver (400×) and quantitative analysis of the relative amount of collagen. Significance was assessed by Student t-test.
Figure 2
Figure 2
Serum lipid profiles and liver function indicators in Control and NAFLD mice. (A) Triglycerides (TG), (B) Total cholesterol (TC), (C) high density lipoprotein (HDL), (D) low density lipoprotein (LDL), (E) alanine aminotransferase (ALT), and (F) aspartate aminotransferase (AST). Significance was assessed by Student t-test.
Figure 3
Figure 3
Alpha diversity analysis of the bacterial community in the jejunum, ileum, cecum, and colon of Control and NAFLD mice. (A) Chao1 index, (B) ACE index, (C) Shannon index, and (D) Simpson index. Significance was assessed by Wilcox Rank-Sum test. *indicate significant difference in the different intestinal segments in the CK group and NAFLD group. #indicates the significant difference in the same intestinal segments between CK group and NAFLD group. *or# p < 0.05; **or## p < 0.01.
Figure 4
Figure 4
Beta diversity analysis of the same intestinal segments between CK group and NAFLD group assessed by NMDS and PCoA based on Bray-Curtis distance. (A,B) Jejunum, (C,D) ileum, (E,F) cecum, and (G,H) colon.
Figure 5
Figure 5
Beta diversity analysis of the jejunum, ileum, cecum, and colon in the same group assessed by NMDS and PCoA based on Bray-Curtis distance. (A,B) CK group, and (C,D) NAFLD group.
Figure 6
Figure 6
Venn diagram based on OTU. (A) The jejunum between CK and NAFLD (NA) groups, (B) the ileum between CK and NAFLD (NA) groups, (C) the cecum between CK and NAFLD (NA) groups, (D) the colon between CK and NAFLD (NA) groups, (E) the jejunum, ileum, cecum, and colon of CK group, and (F) the jejunum, ileum, cecum, and colon of NAFLD (NA) group.
Figure 7
Figure 7
Relative abundance of bacteria at phylum level. (A) Stacked column graph of the top 10 phyla with the highest relative abundance. (B) The box diagram was used to analyze the differences in relative abundance of the five phyla with the highest relative abundance. Wilcox Rank-sum test was used to compare the significance of the same intestinal segment between the CK group and the NAFLD group; Kruskal-wallis test was used to compare the differences between different intestinal segments in the same group. *indicate significant difference in the different intestinal segments in the CK group and NAFLD group. #indicates the significant difference in the same intestinal segments between CK group and NAFLD group. *or# p < 0.05; **or## p < 0.01.
Figure 8
Figure 8
Relative abundance of bacteria at genus level. (A) Stacked column graph of the top 10 genera with the highest relative abundance. (B) The box diagram was used to analyze the differences in relative abundance of the five genera with the highest relative abundance. Wilcox Rank-sum test was used to compare the significance of the same intestinal segment between the CK group and the NAFLD group; Kruskal-wallis test was used to compare the differences between different intestinal segments in the same group. *indicate significant difference in the different intestinal segments in the CK group and NAFLD group. #indicates the significant difference in the same intestinal segments between CK group and NAFLD group. *or# p < 0.05; **or## p < 0.01.
Figure 9
Figure 9
Linear discriminant analysis effect size (LEfSe) analysis of microbiota among different intestinal segments in the same group. (A) CK group, and (B) NAFLD (NA) group (α = 0.05, logarithmic Linear Discriminant Analysis (LDA) score threshold = 4.0).
Figure 10
Figure 10
LEfSe analysis of intestinal microbiota in the same segment between CK group and NAFLD (NA) group. (A) Jejunum; (B) Ileum; (C) Cecum; (D) Colon (α = 0.05, logarithmic Linear Discriminant Analysis (LDA) score threshold = 4.0).
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