P2.P319: Linking Ferroptosis and Necrotizing Enterocolitis: Understanding How This Newly Discovered Cell Death Pathway Could Become a Novel Therapeutic Target

Background: Necrotizing enterocolitis (NEC) is a devastating gastrointestinal disease primarily affecting premature infants. Its pathogenesis involves a complex interplay of various factors, including inflammation, hypoxia, and oxidative stress. Recent studies have shed light on the potential role of ferroptosis, a newly identified form of regulated cell death characterized by iron-dependent lipid peroxidation, in the development of NEC. The delicate balance between oxidative stress and antioxidant defense mechanisms in the immature intestine of preterm infants may render them particularly susceptible to ferroptosis. In this study, we hypothesize that ferroptosis plays an important role in NEC pathogenesis. To substantiate this hypothesis, we undertook experiments to assess the expression of crucial ferroptosis regulators within a murine NEC model.

Methods: NEC was induced in five-day-old mouse pups (n=8) using gavage formula feeds and intermittent hypoxia and hypothermia [Indiana University School of Medicine IACUC protocol #22107]. These were compared to breast fed controls (n=8) who remained with their mother. All pups were sacrificed on postnatal day 9. The terminal ileum was isolated from the pups for RNA extraction. RNA quality and quantity was confirmed via spectrophotometer, and cDNA was synthesized from these samples. Using these cDNA samples, real time quantitative polymerase chain reaction (qRT-PCR) was performed to determine the expression levels of ACSL4 and SLC7A11, two critical modulators in the ferroptosis pathway. Using the delta-delta Ct method, qRT-PCR data was used to quantify relative gene expressions. A Mann-Whitney test was used to compare groups. P < 0.05 was considered significant.

Results: In the experimental NEC group, the expression of the ACSL4 gene was found to be significantly downregulated compared to breast-fed controls (p=0.022). Additionally, the expression of the SLC7A11 gene was observed to be downregulated in the experimental NEC group, although this downregulation did not reach statistical significance (p=0.059) (Figure 1).

Conclusion: This study reveals a statistically significant downregulation of the key ferroptosis enzyme ACSL4 in mice pups subjected to experimental NEC, indicating a potential link between NEC and the regulation of this gene. Additionally, the expression of the SLC7A11 gene was observed to be downregulated in the experimental NEC group. Although this did not reach statistical significance, this trend warrants further investigation as it highlights potential dysregulation in the ferroptosis pathway in the context of NEC. Understanding the intricate relationship between ferroptosis and NEC holds promise for the development of innovative therapeutic strategies aimed at mitigating the devastating effects of NEC in vulnerable neonates. Future investigations should assess key ferroptosis protein expression, lipid peroxidation levels, and iron quantities to thoroughly characterize the connection between NEC and ferroptosis. This comprehensive analysis is poised to reveal potential targets for therapeutic intervention, offering new avenues for treatment development.