B. J. Blackwood1,3, D. Wood2, C. Yuan2, J. Nicolas2, C. J. Hunter1,2 1Ann & Robert H. Children’s Hospital Of Chicago,Pediatric Surgery,Chicago, IL, USA 2Northwestern University,Surgery,Chicago, IL, USA 3Rush University Medical Center,Chicago, IL, USA
Introduction: Necrotizing Enterocolitis (NEC) is the most common gastrointestinal emergency of premature infants. Cronobacter sakazakii (CS) has been associated with out breaks of infant NEC. Protein kinase A is a common mediator implicated in cell survival and apoptotic pathways. We hypothesized that protein kinase A (PKA) inhibition would be protective against experimental NEC.
Methods: Rat intestinal epithelial cells (IEC-6) were grown to confluence and exposed to CS. PKA inhibitors (KT-5720 & SC-3010) were added at doses of 0.1uM, 1uM and 10uM prior to CS infection. IEC-6 cell apoptosis was assayed by western blot of Caspase 3, and by TUNEL staining using the ApoTag red kit. PKA siRNA knocked down PKA in IEC-6 cells and apoptosis was assayed. Rat pups were orally administered a PKA inhibitor (KT-5720), prior to induction of experimental NEC with CS and hypoxia. Mortality rates were measured. Intestinal segments were processed for histology, intestinal injury scoring and ApoTag staining, as well as for western blot analysis for caspase-3. Differences were analyzed with ANOVA where appropriate
Results: PKA is present in IEC-6 and appears to be activated by infection with CS. The addition of a PKA inhibitor prior to IEC-6 infection with CS prevents CS-induced apoptosis (p<0.005). Western Blot analysis revealed a five-fold reduction in expression of PKA in IEC-6 cells with PKA siRNA knockdown. Additionally, a four-fold reduction in Caspase 3 expression was seen in these same cells. Rat pups given the PKA inhibitor prior to induction of experimental NEC, has significantly lower mortality (FIgure 1) (p < 0.001), less intestinal injury (p< 0.001), and decreased intestinal apoptosis as compared with controls.
Conclusion: We conclude that PKA mediated signaling may play an important role in CS-induced intestinal epithelial apoptosis, and that PKA inhibition is protective against experimental NEC. The prospect of PKA inhibitors presents an interesting potential therapeutic line of investigation.
