A. A. Golubkova1, T. A. Leiva1, H. A. Liebe1, K. B. Snyder1, C. A. Schlegel1, J. M. Hansen3, P. F. Vitiello2, C. J. Hunter1 1University Of Oklahoma Health Sciences Center, Department Of Pediatric Surgery, Oklahoma City, OK, USA 2University Of Oklahoma Health Sciences Center, Neonatal-Perinatal Medicine, Oklahoma City, OK, USA 3Brigham Young University College of Life Sciences, Department Of Cell Biology And Physiology, Provo, UTAH, USA
Introduction: Necrotizing enterocolitis (NEC) is an intestinal disease that primarily affects premature infants. Among the mechanisms that determine disease severity, immature molecular response and defense systems at the intestinal epithelial cell (IEC) are implicated. Oxidative stress has a key role in dysregulated cellular injury, but the function of glutathione antioxidant system in response to oxidative stress in NEC is yet to be fully described. We hypothesized that intestinal tissue affected by NEC will show evidence of oxidized glutathione redox potentials and a specific player of this antioxidant system, glutathione peroxidase 4, is deregulated at baseline or in response to NEC stress in premature infants.
Methods: Human intestinal specimens were collected from infants undergoing resection for surgical NEC or other clinically indicated reason (i.e., ostomy takedown). Tissue was analyzed via high-performance liquid chromatography to determine glutathione (GSH) and glutathione disulfide (GSSG) concentrations and derive GSH redox potentials. GPX4 expression from extracted protein was compared by Western blot between NEC versus non-NEC tissue. Human enteroids derived from intestinal crypts with history of NEC versus non-NEC were cultured and then subjected to 12 hours of LPS and hypoxia to induce NEC in a laboratory setting. Changes in GPX4 mRNA expression were determined via qRT-PCR to compare between enteroids of different origin as well as in response to experimental NEC induction.
Results: Intestinal tissue with active NEC shows significantly decreased GSH, increased GSSG concentrations, and consequently significantly oxidized GSH redox potentials (p-value <0.05). GPX4 protein expression is lower in specimens with NEC (Figure 1, p-value <0.05). A reduction in mRNA expression of GPX4 is also evident with induction of experimental NEC in enteroids.
Conclusions: Oxidative stress is evident in NEC. The pathways through which it leads to injury involve a variety of mechanisms, of which the role of the glutathione antioxidant system deserves attention. We show that GPX4 is downregulated at both gene and protein level in response to NEC, in human intestinal tissue with active NEC and in an in vitro enteroid NEC model. GPX4 is a unique member of its family as it is capable of reducing lipid radicals. Downregulated or dysfunctional GPX4 may lead to toxic build up of oxidized fatty acids and tip a cell towards regulated cell death. Investigation into the response of this enzyme to NEC and its downstream effects will further define the role of oxidative stress and the glutathione antioxidant system in the timeline of cellular injury in NEC.
