Antimicrobial Effects of Kelisha Capsule on Escherichia coli Induced Diarrhea in Mice

Abstract

Background: Kelisha capsule (KLSC), listed in the Chinese Pharmacopoeia, has been employed for the treatment of infectious diarrhea. Nevertheless, the precise mechanism of KLSC remains to be elucidated.

Aim of the study: This work was to investigate the antibacterial mode and therapeutic mechanism of KLSC towards E. coli infected diarrhea.

Materials and methods: HPLC identified the primary chemical constituents of KLSC. A model of diarrhea was induced via E. coli injection. The impact of KLSC on E. coli-induced diarrhea was evaluated using a diarrhea score in Babl/c mice. The integrity of the intestinal barrier was assessed through staining and measuring levels of specific proteins. Furthermore, immunofluorescence staining was conducted to identify tight junction proteins in the small intestinal tissue. The full-length 16S rRNA was used to examine gut microbiota. Network pharmacology, molecular docking, and molecular dynamic simulation were used to investigate the impact of KLSC on diarrhea-related inflammation and quantify the expression levels of IL-6 and TNF-α in the blood and small intestine. The in vivo antibacterial activity and mode of action of KLSC were also investigated using IVIS imaging, transmission electron microscopy, scanning electron microscopy, and molecular dynamic simulation.

Results: KLSC exhibited a positive effect on E. coli infected diarrhea. The content of IL-6 and TNF-α in mice with KLSC was significantly reduced. Additionally, KLSC could restore the intestinal barrier function by reversing small intestine structure and up-regulating the expression of tight junction proteins. The KLSC significantly inhibit pathogenic bacteria and restore the gut microbiota diversity. IVIS Imaging System was visually observed significant antibacterial efficacy of KLSC in vivo. The KLSC disrupted the cell membrane system of E. coli through the interaction between bioactive molecule and bilayer.

Conclusion: KLSC normalized the gut barrier function, reshaped gut microbiota balance, suppressed the inflammatory pathways, and inhibited the bacterial activity, thereby relieving the diarrhea caused by E. coli.

Keywords: Kelisha capsule; antibacterial mechanism; diarrhea; gut microbiota; molecular dynamics simulation.

Graphical abstract

Figure 1

KLSC demonstrated the protective effect in mice with diarrhea. (A) The diagram illustrates the experimental steps. (B) The changes in diarrhea score. (C) The images of small intestine tissues stained with HE. (D) Villus lengths of various groups.

Figure 2

The in silico predictive outcomes of KLSC. (A) Prediction of key compounds of KLSC. (B) PPI network analysis. (C) Molecular docking of key ingredients and key targets. (D) Molecular dynamics simulation of key ingredients and key targets. (E) The contents of inflammatory factors in the serum. (F) The contents of inflammatory factors in the small intestine tissue.

Figure 3

KLSC maintained small intestinal mucosal barrier function. (A) the contents of DAO and ITF. (B) Immunofluorescence analysis of tight junction proteins. (C) Quantitative analysis of ZO-1 and occludin expression levels.

Figure 4

The structure and diversity of the gut microbiota in different groups. (A) Rarefaction curves. (B) Shamon index. (C) Chao index. (D) Microbial dysbiosis index. (E) PCoA score plots. (F) NMDS score plots.

Figure 5

KLSC regulated compositions of gut microbiota. (A) Bar plot of community composition at the phylum level. (B) bar plot of community composition at species level. (C) Correlation analysis between microbiota and biochemically diarrhea traits. (D–F) Abundance changes of Escherichia coli, Enterococcus faecalis, Ligilactobacillus murinus.

Figure 6

The antibacterial activity of KLSC in vivo and in vitro. (A) Monitoring the bioluminescent signal of Escherichia coli. (B) The images of SEM and TEM in control group. (C) The images of SEM and TEM in KLSC group. (D) Conformations of membrane model in different systems. (E) Density profiles of the bilayer. (F) Hydrogen bond numbers. (G) The surface area of the lipid bilayer.