Guolin Shi,1,2 Xiao Lu,2 Guowei Wang,1 Kai Qian,2 Xiaokang Peng,2 Yangyang Zhu,2 Jiaxu Wu,2 Haiyong Ke,2 Li Chen,2 Yuanhang Zheng,2 Tao Yang,2 Xiaoying Shi,2 Pintong Huang1,3,4 

1Department of Ultrasound in Medicine, The Second Affiliated Hospital of Zhejiang University School of Medicine, Zhejiang University, Hangzhou, 310009, People’s Republic of China; 2Post-Doctoral Research Center, Zhejiang SUKEAN Pharmaceutical Co., Ltd, Hangzhou, 311228, People’s Republic of China; 3Research Center of Ultrasound in Medicine and Biomedical Engineering, The Second Affiliated Hospital of Zhejiang University School of Medicine, Zhejiang University, Hangzhou, 310009, People’s Republic of China; 4Research Center for Life Science and Human Health, Binjiang Institute of Zhejiang University, Hangzhou, 310009, People’s Republic of China

Correspondence: Pintong Huang, The Second Affiliated Hospital of Zhejiang University School of Medicine, Zhejiang University, 88 Jiefang Road, Shangcheng District, Hangzhou, 310009, People’s Republic of China, Email huangpintong@zju.edu.cn

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, diarrhea, antibacterial mechanism, molecular dynamics simulation, gut microbiota