Xin-Jia-Tong-Xie-Yao-Fang restores the intestinal barrier to alleviate irritable bowel syndrome via microbial butyrate mediated PI3K/Akt pathway suppression

BACKGROUND: Xin-Jia-Tong-Xie-Yao-Fang (XJTXYF) is a proven prescription for managing diarrhea-predominant irritable bowel syndrome (IBS-D), which is a prevalent functional gastrointestinal disorder. However, the underlying mechanism of XJTXYF remains unclear. This study aims to explore the material basis and potential mechanisms of XJTXYF against IBS-D. METHODS: The constituents of XJTXYF were identified by UPLC-Q-TOF-MS/MS, and potential targets and pathways were screened by network pharmacology. Changes in the intestinal microbiome and fecal butyric acid levels after XJTXYF administration were analyzed through 16S rRNA sequencing and HPLC respectively. The abdominal withdrawal reflex score, serum levels of FITC-D and the expression of intestinal epithelial tight junction proteins, as well as the activity of the PI3K/Akt pathway were assessed. In-vitro experiments involved silencing GPR109A using small interfering RNA and inducing barrier damage with LPS pre-treatment in the Caco-2 cell line. Barrier function was evaluated by FITC-D permeability and tight junction protein expressions. The activity of the PI3K/Akt pathway was determined through Western blotting analysis. RESULTS: The major active compounds of XJTXYF identified by UPLC-Q-TOF-MS/MS include Saikosaponin C, Hesperetin, Neohesperidin, Albiflorin, Quercetin and so on. The PI3K/Akt signaling pathway was predicted by network pharmacology as a potential target of XJTXYF in treating IBS-D. Animal experiments demonstrated that XJTXYF could increase body weight, improve visceral hypersensitivity, rebalance gut flora dysbiosis and upregulate fecal butyric acid content in IBS mice. The XJTXYF also showed the abilities of intestinal barrier repairment and PI3K/Akt pathway suppression. Fecal microbiota transplantation and the positive controls (NaB and Clostridium Butyricum) verified that a causal relationship existed between gut microbiome changes and IBS-D improvement after XJTXYF treatment. In-vitro experiments revealed the role of butyrate in barrier protection via PI3K/Akt inhibition. CONCLUSION: Overall, in this study, we revealed that XJTXYF could restore intestinal barrier function through microbial butyrate mediated PI3K/Akt inhibition and improve IBS-D symptoms.