Tong-Xie-Yao-Fang Modulates Mucosal Repair via CRH-TLR4- Mediated Autophagy in Stress-Induced Colitis

Background: Autophagy plays an essential role in inflammatory processes and mucosal repair, with Corticotropin-Releasing Hormone (CRH) and Toll-Like Receptor 4 (TLR4) also playing significant roles in regulating cellular autophagy. We hypothesized that disruptions in the CRHR2-mediated CRH-TLR4 signaling axis are associated with altered autophagy in intestinal epithelial cells (IECs) in ulcerative colitis. Furthermore, we investigated how these disruptions impact the mucosal repair mechanisms that are enhanced by Tong-Xie-Yao-Fang(TXYF). Methods: A 3% dextran sodium sulfate (DSS) solution was administered to mice for five days to induce a colitis model. Restraint stress was applied for 2 hours daily. while the CRHR2 antagonist Astressin 2B (Ast2B) at a dose of 20 μg/(kg·day) was given intraperitoneally, the CRHR2 agonist UCN2 at 30 μg/(kg·day) was administered peritoneally. TXYF at 5.6 g/(kg·day) was delivered by gavage, all for nine consecutive days. Body weight, fecal characteristics, and occult blood were monitored across all groups of mice throughout the experiment. The mice were euthanized by cervical dislocation, and the entire colon distal to the ileum was immediately dissected for further analysis. This included histological scoring of the colon, ELISA to detect changes in CRH and TLR4 levels in the colonic mucosa, Western blot analysis to assess autophagy through the LC3II/LC3I ratio and P62 levels, and in situ detection of apoptosis in intestinal epithelial cells using the TUNEL method. Results: Compared to the DSS group, DSS mice subjected to daily restraint stress exhibited increased TLR4 expression (P=0.0241<0.05), greater weight loss (P=0.0343<0.05), higher histological scores (P=0.0171<0.05), and an elevated apoptosis index (P=0.0375<0.05). Additionally, these mice had higher serum IL-6 levels than those given DSS alone (P=0.0004<0.05). The DSS+Ast2B group showed a higher LC3BⅡ/LC3BⅠ ratio compared to the DSS+NS group (P=0.0417<0.05). In contrast, the DSS+Ucn2 group had lower LC3BⅡ/LC3BⅠ (P=0.0011<0.05) and TLR4 (P=0.0025<0.05) protein expression, along with higher p62 protein levels (P=0.0129<0.05) compared to the DSS+NS group. Correlation analysis revealed a significant association between TLR4 and autophagy-related proteins (LC3 and p62) (P<0.05). Compared to the DSS+STRESS+NS group, the DSS+STRESS+TXYF group showed lower expression of CRH (P=0.0031<0.05) and TLR4 (P=0.0058<0.05), with reduced autophagy levels (all P<0.05). Additionally, the DSS+STRESS+TXYF group demonstrated increased colon length (P=0.0004<0.05) and reduced DAI scores (P=0.0384<0.05), histopathological scores (P=0.0015<0.05), and apoptosis levels (P=0.0125<0.05). Compared to the DSS+STRESS+Ast2B group, the DSS+STRESS+TXYF+Ast2B group showed lower expression of CRH (P=0.0109<0.05) and TLR4 (P=0.0057<0.05), along with reduced levels of the autophagy-related protein LC3 (P=0.0146<0.05). Furthermore, the DSS+STRESS+TXYF+Ast2B group exhibited increased colon length (P<0.0001) and reduced DAI scores (P<0.0001), histopathological scores (P<0.0001), and apoptosis levels (P=0.0002<0.05). Conclusion: Restraint stress enhances the expression of TLR4, thereby exacerbating colitis. CRHR2 serves to inhibit TLR4 protein expression, consequently reducing autophagy levels in intestinal epithelial cells. TXYF mediates the effects of CRHR2 on the CRH-TLR4 signaling axis, diminishing apoptosis in intestinal epithelial cells, inhibiting autophagy, preserving the integrity of the intestinal mucosal barrier, and exerting anti-inflammatory effects.