Tong-Xie-Yao-Fang alleviates diarrhea-predominant irritable bowel syndrome in rats via the GCN2/PERK-eIF2α-ATF4 signaling pathway

BACKGROUND: Diarrhea-predominant irritable bowel syndrome (IBS-D) is a common functional gastrointestinal disease. Tong-Xie-Yao-Fang (TXYF), the traditional Chinese herbal medicine prescription, is a classic and effective prescription for the treatment of IBS-D, but its mechanism of action is not fully clarified. OBJECTIVE: To evaluate the efficacy of TXYF in the treatment of IBS-D and to explore its potential mechanism of action. METHODS: Changes in the serum levels of 50 free amino acids were targeted for detection by high-performance liquid chromatography (HPLC), and the expression of glucose-regulated protein 78 (GRP78), general control nonderepressible 2 (GCN2), and endoplasmic reticulum-resident kinase (PERK) was detected by immunohistochemistry examinations in healthy volunteers and IBS-D patients. The IBS-D rat was constructed by the three-factor superposition method of neonatal maternal separation, 2,4,6-trinitrobenzene sulfonic acid enema, and chronic unpredictable stress stimulation. The treatment effect of TXYF on IBS-D rats was observed by recording the body weight, grasp force, fecal water content (FWC), and abdominal withdrawal reflex (AWR) of rats before and after treatment. The effects of GCN2/PERK-eukaryotic initiation factor-2 (eIF2α) -activating transcription Factor 4 (ATF4) pathway proteins and gene expression were analyzed by western blotting, reverse transcription-polymerase chain reaction (RT-qPCR), and immunohistochemistry evaluations. RESULTS: Compared with healthy volunteers, IBS-D patients exhibited lower levels of cysteine, γ-aminoacetic acid (GABA), homoproline, and lysine, and immunohistochemistry showed strong activation of GRP78, a marker of endoplasmic reticulum stress. Differential expression of GCN2 and PERK proteins was detected in IBS-D patients and rat colons. In the IBS-D rats, TXYF improved the body weight and grasp force, reduced the FWC, and improved the AWR score. TXYF increased the levels of p-GCN2 and GCN2 and reduced the levels of GRP78, p-PERK, PERK, p-eIF2α, and eIF2α, thereby affecting the expression of the apoptosis-related transcription factors ATF4, CHOP, Caspase-3, and Bcl-2. CONCLUSION: Our study showed that TXYF improved IBS-D by inhibiting apoptosis. The anti-apoptosis effects were potentially mediated by regulating the GCN2/PERK-eIF2a-ATF4 signaling pathway.