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

UPLC-Q-TOF-MS/MS identified major active compounds of XJTXYF including Saikosaponin C, Hesperetin, Neohesperidin, Albiflorin, and Quercetin.

• Constituents of XJTXYF were identified using UPLC-Q-TOF-MS/MS methodology.
• Multiple bioactive compounds were characterized from this traditional Chinese medicine prescription.
• Network pharmacology was used to screen potential targets and pathways based on the identified compounds.

Network pharmacology analysis predicted the PI3K/Akt signaling pathway as a potential target of XJTXYF in treating IBS-D.

• Network pharmacology approach was used to screen potential targets and pathways from XJTXYF compounds.
• The PI3K/Akt pathway was identified as a key predicted mechanism of action.
• This prediction was subsequently validated through both in vivo and in vitro experiments.

XJTXYF treatment improved multiple IBS-D symptoms in animal models including body weight, visceral hypersensitivity, gut flora dysbiosis, and fecal butyric acid content.

• XJTXYF increased body weight in IBS mice.
• Visceral hypersensitivity was assessed using abdominal withdrawal reflex scores.
• Gut flora dysbiosis was rebalanced after XJTXYF administration as analyzed through 16S rRNA sequencing.
• Fecal butyric acid content was upregulated after XJTXYF treatment, measured by HPLC.

XJTXYF demonstrated intestinal barrier repair and PI3K/Akt pathway suppression in IBS animal models.

• Intestinal barrier function was assessed via serum FITC-D levels and expression of intestinal epithelial tight junction proteins.
• PI3K/Akt pathway activity was determined through Western blotting analysis.
• XJTXYF showed abilities of intestinal barrier repairment concurrent with PI3K/Akt pathway suppression.

Fecal microbiota transplantation experiments confirmed a causal relationship between gut microbiome changes and IBS-D improvement after XJTXYF treatment.

• Fecal microbiota transplantation was used to verify causality between microbiome changes and therapeutic effects.
• Positive controls included sodium butyrate (NaB) and Clostridium butyricum.
• These experiments verified that gut microbiome changes caused by XJTXYF were causally linked to IBS-D improvement.

In vitro experiments using Caco-2 cells revealed that butyrate protects intestinal barrier function via PI3K/Akt pathway inhibition.

• Caco-2 cell line was used with LPS pre-treatment to induce barrier damage.
• GPR109A was silenced using small interfering RNA to investigate the receptor's role in butyrate signaling.
• Barrier function was evaluated by FITC-D permeability and tight junction protein expressions.
• PI3K/Akt pathway activity was determined through Western blotting analysis.
• Results revealed butyrate's role in barrier protection specifically via PI3K/Akt inhibition.