Protective Effect of Lactiplantibacillus plantarum 1201 Combined with Galactooligosaccharide on Carbon Tetrachloride-Induced Acute Liver Injury in Mice

Abstract

Acute liver injury (ALI) has a high mortality rate of approximately 20-40%, and it is imperative to find complementary and alternative drugs for treating ALI. A carbon tetrachloride (CCl₄)-induced ALI mouse model was established to explore whether dietary intervention can alleviate ALI in mice. Intestinal flora, intestinal integrity, biomarkers of hepatic function, systemic inflammation, autophagy, and apoptosis signals were detected through a real-time PCR, hematoxylin-eosin staining, 16S rRNA gene sequencing, and so on. The results showed that Lactiplantibacillus plantarum 1201 had a strongly antioxidant ability, and galactooligosaccharide (GOS) could boost its growth. Based on these findings, the combination of L. plantarum 1201 and GOS, the synbiotic, was applied to prevent CCl₄-induced ALI in mice. The current research proved that GOS promoted the intestinal colonization of L. plantarum 1201, and the synbiotic improved the antioxidant capacity of the host, regulated the intestinal flora, repaired the intestinal barrier, inhibited the activation of the MAPK/NF-κB pathway, and then inhibited the apoptosis and autophagy pathways, relieving inflammation and liver oxidation; thereby, the ALI of mice was alleviated. These results suggest that synbiotics may become a new research direction for liver-protecting drugs.

Keywords: Lactobacillus plantarum 1201; acute liver injury; galactooligosaccharides; intestinal flora; synbiotic.

Figure 1

GOS promoted the growth of L. plantarum 1201 with high-quality antioxidant power. (A) Growth curve of L. plantarum 1201 in GOS treatment mediums and the MRS medium. (B) Picrylhydrazyl free radical (DPPH) radical scavenging assay (%) of L. plantarum 1201. (C) Hydrogen peroxide capacity (%) on L. plantarum 1201. (D) Reducing power concentration on L. plantarum 1201. CFS, cell-free supernatants group; CS, cell supernatants group; CFE, cell-free extracts group; PC, Positive control group. * p < 0.05; ** p < 0.01; *** p < 0.001; * is compared with C in Figure 1A; paired two-tailed t-test.

Figure 2

Synbiotics alleviated the dysfunction and pathological damage of the liver in CCl₄-induced ALI mice. (A) Experimental set-up: C57BL/6N mice with an intraperitoneal injection of 2 μL/g 50% CCl₄ peanut oil solution treated with probiotics, prebiotics, or synbiotics by daily gavage. (B) Liver index (n = 9 mice/group; each data point represents one mouse). (C,D) ALT and AST enzyme activity in mouse serum (n = 3). (E) H&E pathological section of mouse liver tissue (n = 4). * p < 0.05; ** p < 0.01; *** p < 0.001; * is compared with MD; paired two-tailed t-test.

Figure 3

Synbiotics reduced oxidative stress and relieved liver inflammation in CCl₄-induced ALI mice. (A–C) Homogenizing the liver tissues to examine the activities of SOD (A), the MDA (B), and GSH content (C) (n = 3). (D) the expression levels of IFN-γ, IL-1β, TNF-α, IL-6 and Chemokines (CCL4, CCL5) (n = 3). (E) the expression levels of anti-inflammatory factors (IL-10, IL-22, TGF-β). (F,G) the mRNA level of Bcl-2 (F) and FN-1 (G) (n = 3). * p < 0.05; ** p < 0.01; *** p < 0.001; * is compared with MD; paired two-tailed t-test.

Figure 4

Synbiotics alleviated intestinal flora disturbance in CCl₄-induced ALI mice. (A) PCoA analysis chart, PC1 vs. PC2, “the dots represent each sample; the horizontal and vertical coordinates are the two characteristic values that cause the largest differences between the samples” (n = 5). (B) Three-dimensional PCoA analysis chart, PC1 vs. PC2 vs. PC3. (C) Rarefaction curve (n = 5). (D) Shannon index (n = 5). (E) Chao index (n = 5). (F) Analysis of Variance between groups at the phylum level. (G) Analysis of Variance between groups at the family level. (H,I) Relative abundance of the gut bacterial composition at the level of the phylum and species (mean relative abundance > 0.1%). (J) Family-level network diagram of each species, “the circle represents the species, the size of the circle represents the average abundance of the species; the line represents the correlation between the two species, the thickness of the line represents the strength of the correlation, and the color of the line: orange represents a positive correlation, and green represents a negative correlation”. * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001; * is compared with MD; paired two-tailed t-test.

Figure 5

Synbiotics relieved intestinal inflammation in CCl₄-induced ALI mice. (A) the expression levels of IFN-γ, IL-1β, TNF-α, IL-6, CCL4, and CCL5 in the colon tissues (n = 3). (B) the expression levels of IL-10, IL-22, and TGF-β in the colon tissues (n = 3). (C) the mRNA level of Bcl-2 (n = 3). (D) the mRNA levels of claudin3, occludin, zonula occludin-1 (n = 3). (E) H&E pathological section of the mouse colon tissue (n = 4). * p < 0.05; ** p < 0.01; *** p < 0.001; * is compared with MD; paired two-tailed t-test.

Figure 6

Synbiotics inhibited the MAPK/NF-κB signaling pathway, then inhibited the apoptotic signaling pathway and the autophagic signaling pathway in CCl₄-induced ALI mice. (A,B) mRNA levels of the MAPK signaling pathway-related genes in the liver (A) and colon (B) (n = 3). (C–E) Quantitative analysis of protein expressions of NF-κB signaling (C), apoptotic signaling (D), and autophagic signaling (E) pathway-related proteins (n = 3–4). * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001; * is compared with MD; paired two-tailed t-test.

Figure 7

Schematic representation of the role of intestinal flora in the improvement of ALI through synbiotics. After intraperitoneal injection, CCl₄ damages the liver, causing oxidative damage and inflammation of the liver; then it affects the balance of intestinal flora through the liver–gut axis, causing intestinal inflammation and damage, and intestinal damage in turn aggravates liver damage, causing autophagy and apoptosis. However, synbiotics act directly on the intestinal flora, relieving intestinal inflammation, inhibiting the autophagy and apoptosis pathways, and protect ing the liver from inflammation and oxidative damage.