A20 inhibites intestinal mucosal permeability of IBS-D rats by regulating STAT3 signaling pathway

Purpose A20 (TNFAIP3) is a ubiquitin-modifying enzyme that plays a central role in regulating inflammatory responses and cell death across various tissues, including the gastrointestinal tract, skin, and lungs. Nevertheless, its function in irritable bowel syndrome with diarrhea (IBS-D), a prevalent gastrointestinal disorder frequently associated with impaired intestinal barrier integrity, remains poorly understood. The present study sought to elucidate the molecular mechanism through which A20 modulates intestinal mucosal permeability in IBS-D. Methods An IBS-D rat model was established via a combined protocol of maternal separation and intracolonic acetic acid instillation. Multiple physiological and biochemical parameters were assessed, including defecation frequency, fecal water content (FWC), total intestinal permeability, and serum or mucosal levels of D-lactic acid, endotoxin, secretory immunoglobulin A (sIgA), and diamine oxidase (DAO). Immunohistochemical analysis was performed to evaluate A20 protein expression in distal colonic tissues. Additionally, enzyme-linked immunosorbent assay (ELISA), real-time quantitative polymerase chain reaction (RT-qPCR), and western blotting were utilized to measure the expression of tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) in colonic mucosa, as well as the expression of A20, Janus kinase 1 (JAK1), signal transducer and activator of transcription 3 (STAT3), phosphorylated JAK1 (p-JAK1), phosphorylated STAT3 (p-STAT3), and the tight junction proteins Claudin-1 and Claudin-2 in intestinal epithelial cells (IECs). Results The IBS-D rat model was successfully validated. Relative to control animals, IBS-D rats displayed a significant increase in total intestinal permeability, along with elevated systemic levels of endotoxin, D-lactic acid, and DAO, whereas sIgA levels were considerably reduced. Furthermore, A20 expression was markedly downregulated in IBS-D rats, accompanied by enhanced activation of the STAT3 signaling pathway. Overexpression of A20 in IECs led to upregulated Claudin-1 expression and concurrent downregulation of STAT3 activation, Claudin-2, TNF-α, and IL-6. Importantly, both A20 overexpression and pharmacological inhibition of the STAT3 pathway resulted in a significant improvement in intestinal mucosal barrier function. Conclusion These findings demonstrate that A20 enhances intestinal barrier integrity in IBS-D rats, an effect mediated, at least in part, through suppression of the STAT3 signaling pathway.