Betaine inhibits Toll-like receptor 4 responses and restores intestinal microbiota in acute liver failure mice

Abstract

Previous research has revealed that the gut microbiome has a marked impact on acute liver failure (ALF). Here, we evaluated the impact of betaine on the gut microbiota composition in an ALF animal model. The potential protective effect of betaine by regulating Toll-like receptor 4 (TLR4) responses was explored as well. Both mouse and cell experiments included normal, model, and betaine groups. The rat small intestinal cell line IEC-18 was used for in vitro experiments. Betaine ameliorated the small intestine tissue and IEC-18 cell damage in the model group by reducing the high expression of TLR4 and MyD88. Furthermore, the intestinal permeability in the model group was improved by enhancing the expression of the (ZO)-1 and occludin tight junction proteins. There were 509 operational taxonomic units (OTUs) that were identified in mouse fecal samples, including 156 core microbiome taxa. Betaine significantly improved the microbial communities, depleted the gut microbiota constituents Coriobacteriaceae, Lachnospiraceae, Enterorhabdus and Coriobacteriales and markedly enriched the taxa Bacteroidaceae, Bacteroides, Parabacteroides and Prevotella in the model group. Betaine effectively improved intestinal injury in ALF by inhibiting the TLR4/MyD88 signaling pathway, improving the intestinal mucosal barrier and maintaining the gut microbiota composition.

Figure 1

Effect of betaine on liver and small intestine tissue pathological changes and serum biochemical indicators in ALF mice. (A) The liver tissues were stained with HE (× 200). (B) The small intestine tissues were stained with HE (× 200). (C–E) The serum levels of ALT, AST, and TBIL in different animal groups. ^#P < 0.05, compared with the normal group; *P < 0.05, compared with the model group. (F–H) The serum levels of TNF-α, IL-1β and IL-18 in each group. (I,J) The relative mRNA levels of TNF-α, IL-1β and IL-18 in liver and small intestine tissue. ^#P < 0.05, compared with the normal group; *P < 0.05, compared with the model group.

Figure 2

Effect of betaine on the TLR4/MyD88 pathway, (ZO)-1 and occludin mRNA levels and intestinal permeability in ALF mice and LPS-stimulated IEC-18 cells. (A) Intestinal permeability in each animal group. (B) The TEER value in different cell groups. (C,D) The mRNA levels of TLR4, MyD88, (ZO)-1 and occludin in different animal groups. (E,F) The mRNA levels of TLR4, MyD88, (ZO)-1 and occludin in different cell groups. ^#P < 0.05, compared with the normal group; *P < 0.05, compared with the model group; ^&P < 0.05, compared with the low-dose betaine group; ^※P < 0.05, compared with the medium-dose betaine group.

Figure 3

Effect of betaine on the TLR4/MyD88 pathway, (ZO)-1 and occludin protein levels in ALF mice and IEC-18 cell induced with LPS. (A–C) The protein levels of TLR4, MyD88, (ZO)-1 and occludin in different animal groups. (D–F) The protein levels of TLR4, MyD88, (ZO)-1 and occludin in different cell groups. ^#P < 0.05, compared with the normal group; *P < 0.05, compared with the model group; ^&P < 0.05, compared with the low-dose betaine group; ^※P < 0.05, compared with the medium-dose betaine group.

Figure 4

OTU analysis. OTU Venn diagram showing different color patterns representing different groups, and the number of overlaps between different color patterns was the number of OTUs shared between the two groups.

Figure 5

Beta diversity, including ANOSIM and PCoA. (A,B) Weighted UniFrac ANOSIM and unweighted UniFrac ANOSIM; (C,D) weighted UniFrac PCoA and unweighted PCoA. (E,F) LEfSe showed a cluster tree (E), and a histogram (F) representing the gut bacteria, which were of important biological significance in each group.

Figure 6

LEfSe showed three heatmaps. (A) The OTUs with a significant difference between different groups. (B) A total of 24 species with significant differences between groups. (C) There were 11 genera that were significantly different between groups.

Figure 7

Species classification and abundance analysis. (A–E) The corresponding histograms of species profiling were produced for each sample at the classification level of phylum, class, order, family, and genus.

Figure 8

Species classification and abundance analysis. (A–E) The corresponding histograms of species profiling were produced for each group at the classification level of phylum, class, order, family, and genus.