Lactobacillus acidophilus and HKL Suspension Alleviates Ulcerative Colitis in Rats by Regulating Gut Microbiota, Suppressing TLR9, and Promoting Metabolism

Abstract

Ulcerative colitis (UC) is a chronic non-specific inflammatory bowel disease with complex pathogenesis. The intestinal flora disturbance affects the homeostasis of the intestinal environment, leading to metabolic imbalance and immune abnormalities of the host, contributing to the perpetuation of intestinal inflammation. We suggest that the combination of anti-inflammatory therapy and the regulation of intestinal flora balance may help in the treatment process. Previously, we used a combination treatment consisting of Lactobacillus acidophilus (Lac) and Chinese medicine Huan Kui Le (HKL) suspension in a UC rat model, where the combined intervention was more effective than either treatment alone. Herein, the mechanism of action of this combined treatment has been investigated using 16S rRNA sequencing, immunohistochemistry, and ELISA methods in the colon, and untargeted metabolomics profiling in serum. Colon protein expression levels of IL-13 and TGF-β were upregulated, whereas those of TLR9 and TLR4 were downregulated, consistent with an anti-inflammatory effect. In addition, gut microbiota structure changed, shown by a decrease in opportunistic pathogens correlated with intestinal inflammation, such as Klebsiella and Escherichia-Shigella, and an increase in beneficial bacteria such as Bifidobacterium. The latter correlated positively with IL-13 and TGF-β and negatively with IFN-γ. Finally, this treatment alleviated the disruption of the metabolic profile observed in UC rats by increasing short-chain fatty acid (SCFA)-producing bacteria in the colonic epithelium. This combination treatment also affected the metabolism of lactic acid, creatine, and glycine and inhibited the growth of Klebsiella. Overall, we suggest that treatment combining probiotics and traditional Chinese medicine is a novel strategy beneficial in UC that acts by modulating gut microbiota and its metabolites, TLR9, and cytokines in different pathways.

Keywords: HKL; Lactobacillus acidophilus; intestinal flora; metabolomics; ulcerative colitis.

FIGURE 1

LH treatment ameliorated inflammatory infiltration and histopathological injury in TNBS-treated rats. (A) Hematoxylin and eosin staining of colon tissue (n = 8). (B) IFN-γ, IL-4, IL-6, IL-10, 1L-12, 1L-13, 1L-17, and TGF-β levels were determined by ELISA (n = 6).**p < 0.01,*p < 0.05 vs. UC group.

FIGURE 2

LH treatment reduced TLR4 and TLR9 expression in vivo. (A) TLR4 immunohistochemical staining in colon tissue. (B) TLR9 immunohistochemical staining in colon tissue. (C) Quantitative determination of immunochemical (IHC) index of TLR4 and TLR9 positive signals. Data are shown as mean + SD (n = 8). **p < 0.01 vs. normal group; ^ΔP<0.05, ^ΔΔP<0.01 vs. UC group; ^■ p < 0.05, ^■■ p < 0.01 vs. LH group.

FIGURE 3

Gut microbiota diversity analysis (n = 5∼6). (A) Sobs and Shannon rarefaction curve of samples. (B) α-Diversity evaluated by Sobs index. (C) α-Diversity evaluated by Ace index. (D) α-Diversity evaluated by Chao index. *p < 0.05, **p < 0.01. (E) NMDS analysis.

FIGURE 4

Gut microbiota composition analysis (n = 5∼6). (A) Bar plot of bacterial richness distribution at the phylum level; (B) bar plot of bacterial richness distribution at the genus level; (C) diagram of Circos analysis. Indicated are the species composition (small left semicircle), the group (color of the outer ribbon), the species (color of the inner ribbon), and the relative abundance of the species in the corresponding samples (length). The large right semicircle represents the distribution of species in samples at the taxonomic level. The outer layer ribbon represents the species, the inner ribbon color represents different groups, and the length represents the distribution proportion of the sample in a specific species.

FIGURE 5

Bar plot of gut microbiota comparison at phylum level of five groups (n = 5∼6). (A) Differential phyla between the UC group and normal group. (B) Differential phyla between the UC group and LH group. (C) Proteobacteria and Verrucomicrobia comparison in the UC and Lac groups. (D) Proteobacteria and Verrucomicrobia comparison in the UC and HKL groups; *0.01<p ≤ 0.05.

FIGURE 6

Bacterial abundance at the genus level (n = 5∼6). Differences between normal and UC (A); UC and LH (B); UC and Lac (C); and UC and HKL (D). (E) Comparison between groups of several bacteria with large change in abundance; (F) Ternary chart in Lac, HKL, and LH groups. (a.bacteroidales_s24-7_group; b.lactobacillus; c.Escherichia-Shigella; d.romboutsia; e.klebsiella; f.pasteurella; g.Turicibacter h.bacteroides; i.Prevotellaceae_ Ga6A1_group; j.Lachnospiraceae_NK4A136_group; k.Ruminococcus_2; l.Blautia; m.(Eubacterium)_xylanophilum; n.Bifidobacterium; o.Prevotellaceae; p.Helicobacter; q.Alloprevotella; r.Prevotellaceae_NK3B31_group; s.Entero coccus; t.Coprococcus_1; u.Lachnoclostridium; v.Christensenellaceae_R-7_group; w.norank_f_Ruminococcaceae; x.Prevotellaceae_Ga6A1_group).

FIGURE 7

LEfSe cladogram (LDA = 3) and LDA bar chart (n = 5∼6). Circular cladogram for niche specialization of microbial compositions in five groups using the LEfSe analysis of the abundance patterns of bacterial taxa. Circles represent taxonomic categories of organisms from the genus level (outermost circle) to the phylum level (innermost circle). Within each given taxon, each small circle represents its lower clade. Yellow nodes indicate no statistically significant differences in a given taxon between the samples of the five groups. The size of the node is proportional to the LDA score. The links (lines) between the nodes mean hypothetical phylogenetic relationships among organisms, which can be traced back to where the lines branch off (hypothetical ancestor).

FIGURE 8

Correlation Heatmaps and network analysis (n = 6). Correlation heatmaps of (A) cytokines and gut microbiota at the phylum level; (B) TLRs and gut microbiota at the phylum level; (C) cytokines and gut microbiota at the genus level; (D) TLRs and gut microbiota at the genus level; (E) network analysis. The figure shows p < 0.05, correlation >0.3, the top 30 species abundance. The size of the nodes indicates the abundance of the species, and different colors indicate different species. The color of the line indicates positive (red) and negative (green) correlation.

FIGURE 9

LH treatment effect on serum metabolism. (A) Scores plot of PCA analysis of all samples; scores plot of PLS-DA of UC vs. normal group (B), HKL group (C), Lac group (D), and LH group (E). The red marks represent UC group samples (n = 7).

FIGURE 10

¹H-NMR spectrum of serum (0.5–10 ppm). (A) Normal; (B) UC; (C) HKL; (D) Lac; and (E) LH. Numbers represent the peaks of metabolites: 1. VLDL; 2. L-Isoleucine, 3. Leucine; 4. Valine; 5.3-hydroxybutyric acid; 6. Lipids; 7. LDL, 8. Lactic acid, 9. Alanine, 10. Lysine; 11. Acetate; 12. Glycoprotein; 13. Methionine; 14. Acetone; 15. Acetoacetic acid; 16. Pyruvic acid; 17. Carnitine; 18. Methylamine; 19. Citric acid; 20. Creatine; 21. Inositol; 22. Glucose; 23. Taurine; 24. Choline; 25. Glycine.

FIGURE 11

Network analysis of the abundances of gut microbiota and serum metabolites.network analysis. The figure shows p < 0.05, correlation >0.5, the top 50 species abundance. The size of the nodes indicates the abundance of the species, and different colors indicate different species. The color of the line indicates positive (red) and negative (green) correlation.

FIGURE 12

Possible partial mechanisms of UC pathogenesis and LH treatment for UC.