Serum TNF-α as an indicator reflecting the severity of intestinal barrier injury in a neonatal rat model of necrotizing enterocolitis

Background and objective: Necrotizing enterocolitis (NEC) is a severe gastrointestinal emergency in preterm infants and is characterized by progressive intestinal barrier injury accompanied by systemic inflammation. This study aimed to determine whether peripheral inflammatory signals reflect structural damage to the intestinal barrier in a graded neonatal rat model of NEC and to identify candidate blood-based indicators of disease severity. Methods: Neonatal Sprague-Dawley rats were assigned to Control, NEC, and NEC + LPS groups. Ileal injury was assessed by histopathology. The localization and expression of the tight junction proteins ZO-1 and Claudin-3 were evaluated by immunohistochemistry, Western blotting, and qPCR. TNF-α and IL-6 levels in ileal tissue and peripheral serum were measured by ELISA. Correlation analysis and leave-one-out cross-validation (LOOCV)-based regression models were used to assess the relationship between peripheral inflammatory markers and tight junction injury. Results: The model showed a clear severity gradient across groups. Histological injury progressively worsened from the NEC group to the NEC + LPS group. In parallel, ZO-1 and Claudin-3 showed progressively disrupted membrane localization and reduced protein and mRNA expression. TNF-α and IL-6 levels in both ileal tissue and serum increased with disease severity. Serum TNF-α and IL-6 were inversely associated with ileal ZO-1 and Claudin-3 expression in complete-case analyses. In exploratory regression analysis, the TNF-α-only model showed higher internal LOOCV performance than the IL-6-only model for estimating both tight junction proteins. Conclusion: NEC progression in neonatal rats was associated with progressive structural disruption of the intestinal barrier and increasing inflammatory activation. Within this experimental framework, serum TNF-α was more closely associated with tight junction injury than IL-6. These findings are preliminary and hypothesis-generating, and serum TNF-α should not yet be considered a validated clinical biomarker before confirmation in larger animal studies, time-course experiments, and clinical cohorts.