Notch Signaling Involved In The Regulation Of Intestinal Barrier Function In Acute And Chronic Pathological Conditions

BackgroundIntestinal barrier is a protective barrier system against the external environment. Insome conditions, such as trauma, burn injury, IBD and TPN, the intestinal barrier wasdestroyed and lead to SIRS and MODS. Studies have been done to explore the mechanismsabout injury and repair of intestinal barrier. However, the precise mechanisms of theseprocesses are still not fully understood.The intestinal epithelium is formed of a single cell layer and it is one of the mostimportant parts of intestinal barrier. Intestinal epithelium supports nutrients and watertransport and maintains a defense against bacteria, intraluminal toxins and antigens. Theintestinal epithelium is one of the most rapidly proliferating tissues in the body. Theintestinal epithelial cells at the top of the villi die and the epithelial cells at the intestinalcrypts proliferate to compensate for the lost cells. This process takes generally4-7d and itis important for the maintenance of epithelial integrity and intestinal homeostasis. In somepathological conditions, the intestinal homeostasis will be destroyed. The intestine is likelythe most sensitive and vulnerable to ischemia/reperfusion injury among the viscera. Inintestinal ischemia/reperfusion (I/R) injury, the intestinal epithelial cells suffer necrosis andapoptosis early after reperfusion and recovery will happen later. Several signaling pathways,such as Wnt、TGF-β、BMP、Hedgehog, were reported to be involved in the regulation ofintestinal epithelial cells proliferation and apoptosis. However, the mechanism is not fullyunderstood. Short bowel syndrome is one of the most regular clinical syndromes which iscaused by excessive small bowel resection. After massive small bowel resection (MSBR),intestinal adaptation will happen. However, the mechanisms of intestinal adaptation needfurther investigation.Studies have shown that Notch signaling played important roles in the maintenance of intestinal epithelium. Notch signaling was involved in the regeneration process of liver, skin,kidney, heart, pancreas and tracheal epithelium. Notch signaling also protects hepatocytesfrom ischemia/reperfusion injury through suppress the apoptosis of hepatocytes. Notchsignaling also plays crucial roles in the intestinal homeostasis. However, in the condition ofintestina ischemia reperfusion and massive small bowel resection the role of Notchsignaling was not understood fully.In this study, we used both animal model and in vitro cell culture model. Real-timePCR, Western blot, RNAi, immunofluorescence and flow cytometry were applied toinvestigate the role of Notch signaling pathway in the intestinal epithelium and therelationship between Notch signaling and intestinal injury.MethodsIntestinal I/R animal model was used, and intestinal tissues were collected.Immunohistochemical examination and TUNEL staining were used to examine theintestinal epithelial cells proliferation and apoptosis. RT-PCR, Western blot andimmunofluorescence technique were applied to investigate the mRNA and proteinexpression of Notch signaling pathway in intestinal epithelium.Then an in vitro IEC-6cells culture model was built to investigate the role of Notchsignaling in the intestinal epithelium. RT-PCR and Western blot were applied to detect themRNA and protein expression of Notch signaling in IEC-6cells suffering from hypoxia.Inhibition of Notch signaling with DAPT and suppression of Notch signaling expressionwith siRNA were performed to examine the relationship between Notch signaling andintestinal epithelial cells proliferation and apoptosis.An MSBR rat model was built and intestinal tissues were collected. The morphologicalchanges of intestinal mucosa were examined. RT-PCR, Western blot and immunohistochemical examination were applied to investigate the mRNA and protein expression ofNotch signaling in intestinal epithelium.An in vitro IEC-6cell model was used to examine the role of Notch signaling inintestinal adaptation. IEC-6cells were given recombinant Notch ligand and mRNA andprotein expression of Notch signaling was investigated. MTT technique was used to detectthe proliferation of IEC-6cells. Results1. The mRNA and protein expression of Jagged2/Notch1/Hes1in intestinal epitheliumsuffering I/R injury increased significantly especially2h after I/R and returned to normallevel6h after I/R. Immunohistochemial results showed that the proteins ofJagged2/Notch1/Hes1were increased and mainly located in the intestinal crypts.Immunohistochemial results of PCNA showed that proliferation of intestinal epithelial cells2h after I/R was increased significantly.2. In in vitro IEC-6culture model, inhibition of Notch signaling with DAPT andsuppression of Jagged-2and Hes-1expression using siRNA significantly inhibited theproliferation of IEC-6cells.3. The mRNA expression of Jagged1/DLL1/Notch2/Hes5and protein expression ofNICD2/Hes5were increased significantly12h after I/R. The TUNEL results showed that I/Rinjury (12h) increased the apoptosis of intestinal epithelial cells significantly.4. In in vitro IEC-6cell model, DAPT and siRNA for Hes5both suppressed the proteinexpression of NICD2and Hes5. Flow cytometric analysis showed that DAPT and siRNAfor Hes5both increased the apoptosis of IEC-6cells suffering from hypoxia.5. An MSBR rat model was built. Morphological results showed that intestinaladaptaion happened3d after MSBR. The mRNA and protein expression of Jagged1/Notch1/Hes1increased significantly in intestinal epithelium after MSBR. Immunohistohemical results showed that the protein of Jagged1/Notch1/Hes1colocated in intestinalcrypt epithelial cells with PCNA.6. Recombinant Jagged1was applied to IEC-6cells. The mRNA and proteinexpression of Notch1and Hes1were increased and proliferation of IEC-6cells was alsoincreased significantly.Conclusion1. The Jagged2/Notch1/Hes1signaling pathway was activated in intestinal epitheliumafter intestinal I/R injury and was involved in intestinal epithelium regeneration throughincreasing the proliferation of intestinal crypt epithelial cells.2. The Notch2/Hes5signaling was activated in intestinal epithelium after intestinal I/Rinjury. The Notch2/Hes5signaling regulates the apoptosis of intestinal epithelial cells afterI/R injury and protects intestinal epithelium from I/R injury.3. The Jagged1/Notch1/Hes1signaling was activated in intestinal epithelium after MSBR and was involved in intestinal adaptation through increasing the proliferation ofintestinal crypt epithelial cells.