Fraxin alleviates intestinal epithelial cell necrosis in ulcerative colitis by inhibiting the P2Y₁₄R/cAMP/PKA/CREB signaling pathway

Qinpi is a classic herb used in traditional Chinese medicine to treat diarrhea and dysentery caused by damp-heat. Modern research has confirmed that it can alleviate inflammation of the colonic mucosa in ulcerative colitis (UC). As its core active component, Fraxin exhibits potent anti-inflammatory and intestinal protective effects; however, the specific molecular mechanisms underlying its treatment of UC remain unclear. It is currently known that intestinal inflammation, epithelial necrosis, and disruption of the mucosal barrier are core pathological features of UC. Abnormal activation of the P2Y₁₄R receptor and the downstream cAMP/PKA/CREB signaling pathway is closely associated with the progression of UC; however, there is a lack of clear experimental evidence to confirm whether Fraxin intervenes in UC by regulating this target and pathway. Accordingly, this study took P2Y₁₄R as the core target and systematically investigated the therapeutic effects and molecular mechanisms of Fraxin on UC using in vivo and in vitro models. This study established an in vivo UC mouse model and an in vitro TSZ-induced HT-29 cell injury model. Using H&E staining, IHC, qPCR, ELISA, Western blot, and immunofluorescence techniques, we examined changes in histopathology, inflammatory factors, tight junction proteins, and signaling pathway protein expression. Molecular docking and CETSA experiments were used to validate the targeted binding relationship between Fraxin and P2Y₁₄R, and a P2Y₁₄R overexpression plasmid was employed to verify the functional specificity of the target. The results indicate that Fraxin significantly alleviates inflammatory damage in UC in both in vivo and in vitro models, suppresses abnormal P2Y₁₄R expression, and that P2Y₁₄R overexpression reverses its therapeutic effects. Concurrently, Fraxin downregulates pro-inflammatory factors such as TNF-α, repairs intestinal tight junctions, and improves mucosal permeability. By enhancing CREB-RIPK1 binding to inhibit RIPK1/MLKL pathway activation, it reduces intestinal epithelial necrosis and effectively improves pathological symptoms and disease indices in UC-affected mice. In summary, Fraxin UC damage by antagonizing P2Y₁₄R, regulating the cAMP/PKA/CREB signaling axis, inhibiting intestinal epithelial necrosis and inflammatory cascades, and repairing the intestinal mucosal barrier. This study elucidated the core mechanism of Fraxin in treating UC, confirmed that the P2Y₁₄R-cAMP/PKA/CREB axis is a key pathway regulating epithelial damage and inflammatory progression in UC, and provided a novel target and experimental basis for targeted natural medicine therapy for UC.