R. M. Rentea1, Y. Guo2, X. Zhu3, M. W. Musch3, D. M. Gourlay4, J. L. Liedel5 1Children’s Mercy Hospital- University Of Missouri Kansas City,Pediatric Surgery,Kansas City, MO, USA 2University Of Chicago,Pediatrics, Section Of Neonatology,Chicago, IL, USA 3University Of Chicago,Medicine,Chicago, IL, USA 4Children’s Hospital Of Wisconsin,Pediatric Surgery,Milwaukee, WI, USA 5Albert Einstein College Of Medicine,Pediatrics And Critical Care,Bronx, NY, USA
Introduction: Necrotizing Enterocolitis (NEC) is a gastrointestinal disease of complex etiology that effects 1 in 10 premature infants. We previously demonstrated that formula feeding inhibits ileal expression of Heat Shock Protein -70 (Hsp70), a critical stress protein within the intestine and that may serve to lower the injury threshold for subsequent stressors. Barrier function for the premature intestine is critical. It is unknown whether decreased neonatal intestinal Hsp70 increases permeability. We sought to determine whether reduced Hsp70 protein expression increases neonatal intestinal permeability.
Methods: Young adult mouse colon cells (YAMC) were utilized to evaluate barrier function (translocation of fluorescent dextran) as well as intestine from Hsp70-/- pups (KO). Sections of intestine were analyzed by Western blot, immunohistochemistry, and real time PCR. Statistical data were analyzed by paired students t-test and expressed as a mean +/- SEM with p<0.05 considered significant.
Results: YAMC cells were sub-lethally heated or treated with expressed milk (EM) to induce Hsp70. Immunostaining demonstrates co-localized Hsp70 and tight junction protein Zona Occludens-1 (ZO-1), suggesting physical interaction to protect tight junction function. The permeability of YAMC monolayers increases following oxidant injury and is partially blocked by Hsp70 induction either by prior heat stress or EM (fluorescent dextran-70 flux oxidant injury 12 µg/mL vs. heat shock 1.5 µg/mL p<0.007 or EM 3.0 µg/mL p<0.01). Consistent with previous results in the rat, Hsp70 was detected in the ileum of all mother fed mouse pups on day 3. RT-PCR analysis demonstrated that the Hsp70 isoforms, 70.1 and 70.3 predominate in WT pup, however, Hsp70.2 predominates in the KO pups via a 3 fold change (p<0.05). While Hsp70 is present in WT milk, it is not present in KO EM (450 ng/mL vs. 0.10 p<0.001).
Conclusion: Hsp70 associates with ZO-1 to maintain epithelial barrier function. Both induction of Hsp70 and exposure to EM prevent stress-induced increased permeability. Hsp70.2 is present in both WT and KO neonatal intestine suggesting a crucial role in epithelial integrity. Induction of the Hsp70.2 isoform appears to be mediated by mother’s milk. These results suggest that mothers milk feeding modulates Hsp70.2 expression and could attenuate injury leading to NEC.