Abstract
Background: Notch signaling has been shown to play an important role in the maintenance of intestinal homeostasis. The purpose of the present study was to investigate the role of Notch signaling in the regulation of intestinal injury in an ischemia reperfusion (I/R) rat model. Method: Male Sprague-Dawley rats were subjected to sham operation or I/R by occlusion of the superior mesenteric artery (SMA) for 30 min. DAPT (a γ-secretase inhibitor, 500μg/100g) or DMSO was given intraperitoneally 2 h before I/R. Intestinal tissue samples were collected at 12 h after reperfusion. The expression of Notch signaling pathway and phosphorylation of STAT were examined by Real-time PCR, Western blot, immunofluorescence and Co-Immunoprecipitation. Apoptosis of intestinal epithelial cells were examined by TUNEL staining. Neutrophil infiltration was assessed by examining myeloperoxidase (MPO) activity in the gut. Finally, an in vitro IEC-6 culture system was further applied to investigate the role of Notch signaling in the regulation of intestinal epithelal apoptosis after I/R injury. Results: In this study, Notch signaling was activated after intestinal I/R injury. The proteins expression of NICD-1 and Hes-5 were increased significantly (NICD-1: 0.35±0.12 vs 0.79±0.27, p<0.05; Hes-5:0.42±0.15 vs 0.93±0.31, p<0.05 ) and immunofluorescence results showed that the proteins were colocalized mainly in the intestinal epithelial cells after I/R injury. Blocking Notch signaling by DAPT led to aggravated intestinal I/R injury, as manifested by deteriorated intestinal barrier function (TER: 78±9.5V.cm2 vs 62±7.3V.cm2), increased inflammation and intestinal epithelial cells apoptosis (18±1.9 cells/100crypts vs 32±2.2 cells/ 100crypts). Notch signaling blockade also resulted in down-regulation of Hes5, leading to reduced formation of the Hes5-STAT3 complex and hypophosphorylation of STAT3 both in vivo and in vitro. Overexpression of a constitutively active STAT3 attenuated I/R injury by reducing apoptosis. Furthermore, IEC-6 cells were protected from I/R injury induced apoptosis after the activation of Notch signaling by ligand stimulation through increased activation of STAT3. Conclusion: Notch signaling was activated and played an important role in the protection of intestine from I/R injury through Hes-5 dependent activation of STAT3.
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