Single and Mixed Strains of Probiotics Reduced Hepatic Fat Accumulation and Inflammation and Altered Gut Microbiome in a Nonalcoholic Steatohepatitis Rat Model

Abstract

As gut dysbiosis has been implicated in the pathogenesis of nonalcoholic steatohepatitis (NASH), probiotic supplementation might be a potential treatment for this condition. The aim of this study was to evaluate the effects of single- and mixed-strain probiotics on the severity of NASH induced by a high-fat, high-fructose (HFHF) diet and their mechanisms of action. Male Sprague-Dawley rats were divided into four groups (n = 7 per group): control group, NASH group, NASH + single-strain group, and NASH + mixed-strain group. In the single-strain and mixed-strain groups, rats received Lactobacillus plantarum B7 and Lactobacillus rhamnosus L34 + Lactobacillus paracasei B13 by oral gavage once daily, respectively. The duration of the study was 6 weeks. Liver tissue was used for histopathology, hepatic fat content was assessed by Oil Red O staining and hepatic free fatty acid (FFA), and hepatic TLR4 and CD14 expression were assessed by immunohistochemistry. Fresh feces was collected for gut microbiota analysis. Liver histology revealed a higher degree of fat accumulation, hepatocyte ballooning, and lobular inflammation in the NASH group, which improved in probiotics-treated groups. The amounts of hepatic fat droplets and hepatic FFA levels were more pronounced in the NASH group than in the control and treatment groups. Serum TNF- α levels were significantly higher in the NASH group than in control and probiotic groups. The expression of CD14 and TLR4 increased in the NASH group as compared with the control and probiotics-treated groups. Alpha diversity was reduced in the NASH group, but increased in both treatment groups. The relative abundance of Lactobacillus significantly decreased in the NASH group, but increased in both treatment groups. The relative abundance of Akkermansia significantly increased in the NASH group, but decreased in both treatment groups. In conclusion, both single-strain and mixed-strain probiotics could improve NASH histology by suppressing inflammatory responses in the liver, with this improvement potentially being associated with changes in the gut microbiota.

Keywords: CD14; TLR4; gut microbiota; nonalcoholic steatohepatitis; probiotics.

Figure 1

Effects of single and mixed strains of probiotics on liver histopathological changes. (A) Control group, (B) NASH group, (C) NASH + single-strain group and (D) NASH+mixed-strain group. Hematoxylin and eosin staining (400×, scale bar 100 µm and n = 7 per group). Yellow arrows indicate steatosis, red arrows indicate lobular inflammation, and blue arrows indicate hepatocyte ballooning.

Figure 2

Effects of single and mixed strains of probiotics on hepatic lipid accumulation. (A–D): Histological images of Oil Red O staining in each group (400×, scale bar 100 µm and n = 7 per group). (E) Bar graph representing the percentage of Oil Red O staining area in each group. (F) Bar graph representing the hepatic free fatty acid levels in each group. Data are expressed as mean ± SEM. * p < 0.05 compared with the control group; ^# p < 0.05 compared with the NASH group; ^$ p < 0.05 compared with the single-strain group.

Figure 3

Effects of single and mixed strains of probiotics on serum tumor necrosis factor-a (A) and interleukin-6 (B). Data are expressed as mean ± SEM. * p < 0.05 compared with the control group; ^# p < 0.05 compared with the NASH group.

Figure 4

Effects of single and mixed strains of probiotics on CD14 expression. (A–D) Representative images of CD14 expression in each group (400×, scale bar 100 µm and n = 7 per group). (E): Bar graph representing the quantitative measurement of CD14 positivity in each group. Black arrows indicate Kupffer cells with brown-stained cytoplasm. Data are expressed as mean ± SEM. * p < 0.05 compared with the control group; ^# p < 0.05 compared with the NASH group.

Figure 5

Effects of single and mixed strains of probiotics on toll-like receptor 4 expression. (A–D) Representative images of TLR4 expression in each group (400×, scale bar 100 µm and n = 7 per group). (E) Bar graph representing the quantitative measurement of TLR4 positivity in each group. Black arrows indicate hepatocytes with brown-stained cytoplasm. Data are expressed as mean ± SEM. * p < 0.05 compared with the control group; ^# p < 0.05 compared with the NASH group.

Figure 6

Effects of single- and mixed-strain probiotics on alpha and beta diversity of gut microbiota (A) Chao1 index; (B) observed species index; (C) Shannon index; (D) Simpson index; (E) principal coordinates analysis (PCoA, based on Bray–Curtis distance).

Figure 7

Effects of single and mixed strains of probiotics on gut microbiota composition. (A) Top 20 relative abundances of gut microbial composition at the genus level in each group; (B) LEfSe analysis among groups (cutoff of LDA score > 3.0, p-value < 0.005 and FDR-adjusted p-value < 0.1); (C) the associations between depleted microbial compositions and clinical factors; (D) the enrichment of bacteria in NASH vs. control and the correlation with clinical parameters (Spearman’s correlation with p < 0.005)”. The blue dot indicates a positive correlation between the clinical parameter on the diagonal axis and the microorganism on the x-axis. The red dot indicates a negative correlation between the clinical parameter on the diagonal axis and the microorganism on the x-axis. The higher the intensity of the dot color, the higher the r-value, as indicated by the color chart below the graph.

Figure 8

Functional prediction of gut microbial community by PICRUSt2 analysis. Significantly enriched KEGG pathways were found between the NASH and probiotics groups (p-value < 0.001).