K. Wang1,2, Z. Sun1, G. Tao1, P. Lin1, J. Dunn1, K. Sylvester1 1Stanford University,Department Of Surgery, School Of Medicine,Palo Alto, CA, USA 2First Hospital of China Medical University,Gastrointestinal Surgery,Shenyang, LIAONING, China
Introduction:
Necrotizing Enterocolitis (NEC) occurs in preterm newborns following gut colonization and the introduction of enteral feeding. Newborn enteral feeding produces multiple short-chain fatty acids (SCFAs) that are absorbed (propionate, acetate) for systemic utilization or metabolized (butyrate) in the gut. Luminal production of SCFAs is essential for normal gut mucosal function, however we have previously noted a developmental stage sensitivity to butyric acid (BA). We hypothesized that enteric utilization of BA would be coupled to mitochondrial oxidative capacity and oxidative stress buffering according to developmental stage of the host.
Methods:
Human intestinal epithelial stem cells (hIESCs) and their differentiated enterocyte progeny (withdrawal of Wnt agonists) were subject to a titrated dose of NaBu (sodium butyrate) for a determination of cell sensitivity. Following treatment with 10mM NaBu for 24h, LDH release assay was used to detect cellular death and Western blot analyses of cellular lysates for metabolic driver proteins. An in vivo model of SCFA induced metabolic challenge was developed through oral (300mM, 20μl/g body weight twice a day for 3 days) administration to 14d and 28d day mice to represent preterm and term newborns respectively. After 72 hours, mouse stool was assayed for enterocyte specific keratin proteins, markers of intestinal epithelial injury. Mitochondria from 14d and 28d mice were isolated for a determination of mitochondrial Oxidative capacity the oxygen consumption and mitochondrial membrane permeability transition (MPT) detection.
Results:
Following treatment with 10mM NaBu for 24h, hIESCs and differentiated enterocytes from a preterm human newborn demonstrated a significant increase in sensitivity compared to a term human newborn by LDH release assay (0.20±0.03 vs 0.08±0.07,p=0.07, hIESCs; 0.34±0.14 vs 0.05±0.03,p=0.03, differentiated cells). AKT and PGC-1α were significantly downregulated by NaBu in both groups. After 72 hours of NaBu exposure, Keratin protein content in the 14d mouse stool significantly increased compared to the untreated group, suggesting a significant increase in intestinal epithelial cell injury. In functional studies, ADP-induced oxygen consumption was found to be significantly lower in the isolated mitochondria from the gut of 14d mice compared to 28d mice (39.8±12.3 vs 143.84±11.3 pmol/s*ml, p=0.015). MPT assay indicated that mitochondrial swelling and membrane permeability were significantly greater in 14d mice compared to 28d mice under identical conditions.
Conclusion:
Premature developmental stage of enterocytes in both human and mouse is associated with increased sensitivity to NaBu exposure. Mitochondrial capacity for oxidative phosphorylation and redox buffering appears to be deficient in less mature enterocytes. These observations are consistent with age dependent metabolic injury pattern that may be applicable to human NEC.?