Nitric oxide pathway modulation by L-arginine, sildenafil, and glycine in experimental neonatal necrotizing enterocolitis

Abstract Necrotizing enterocolitis (NEC) is a severe inflammatory disease of the immature intestine involving complex interactions between intestinal inflammation, microvascular dysfunction, and innate immune activation. Therapeutic strategies targeting nitric oxide-related pathways and Toll-like receptor (TLR) signaling have been proposed, although their effects remain incompletely understood. This study evaluated the effects of L-arginine, sildenafil, and glycine, administered alone or in combination, in an experimental model of NEC. Ninety-two neonatal Wistar rats were allocated into nine groups: negative control (Breastfeeding group), positive control (NEC induction), and intervention groups receiving oral glycine (10 mg), L-arginine (10 mg), sildenafil (0.3 mg), or their combinations. NEC was induced by formula feeding, hypoxia, and hypothermia. Outcomes included histological intestinal injury, mortality, body weight variation, and TLR4 and TLR9 gene expression assessed by Quantitative Reverse Transcription Polymerase Chain Reaction (RT-qPCR). Overall mortality following NEC induction was 17%. Histological injury scores differed significantly among groups ( p < 0.001), with the highest mean scores observed in the L-arginine monotherapy and glycine plus sildenafil groups. TLR4 expression did not differ significantly among groups ( p = 0.236) but showed a moderate positive correlation with histological severity (r = 0.42). TLR9 expression differed among treatment groups but showed a weak correlation with histological injury (r = 0.18). Treatments targeting nitric oxide-related pathways and inflammatory signaling produced heterogeneous effects in experimental NEC. Glycine monotherapy was associated with lower histological injury scores, whereas L-arginine monotherapy was associated with higher mortality and greater histological severity. These findings suggest that modulation of individual therapeutic targets alone may not consistently reduce intestinal injury in experimental NEC. Given the limitations of the experimental model and the absence of direct assessment of nitric oxide pathway activity, these findings should be interpreted with caution and require confirmation in future studies.