The Role Of Hedgehog Signalling In The Repair Of Small Intestinal Epithelium

?Objective?Gastrointestinal injury and damage to these structures is common in many diseases, as well as after cytotoxic chemotherapy. Given time, the intestines usually recover from such injury, but the underlying molecular mechanisms that govern this repair are largely unknown. As recapitulation of embryological developmental pathways guides injury repair in many tissues, Hedgehog signalling, a conserved and essential embryological pathway in gastrointestinal tract development, may be involved in the repair of small intestinal epithelial damage. Recent studies have demonstrated hedgehog signalling in the adult small intestine, where it is involved in the homeostatic control of epithelium production.In this project, we aimed to establish a gut injury model by 5-flurouracil (5-FU), a chemotherapy drug, and assess the repair process after down regulating shh signalling by inhibitor as well as when upregulating shh signalling by gene modification. ?Methods?A mouse model of 5-FU induced injury in the small intestine was optimised. Assessment and quantification of the affects of injury on morphology were undertaken through hemotoxylin and eosin (H&E) staining, markers of proliferation (proliferatin cell nuclear antigen, PCNA), mitosis (p Histon3) and apoptosis (cleaved caspase3) were shown with immunohistochemistry. The injury and repair of small intestinal epithelium was characterised, and Hedgehog expression during these processes was shown using immunohistochemistry and in situ hybridization. The real time PCR of genes in Hh pathway was also applied. The activity of?-galactosidase in intestine of Ptch/LacZ mice was observed after 5-FU treatment. Differentiated cells were quantified by periodic acid-schiff stain, immunohistochemistry of Lysozyme and synaptophysin responsed to goblet cells, paneth cells and enteroendocrine cells respectively. This injury model was combined with the hedgehog signalling inhibitor, and tissue response in the repairing small intestinal epithelium was assessed using the above techniques. Dual immunofluorescence staining of A33 and GFP was carried out on the intestine of Lgr5-GFP mice, whose intestinal stem cells expressed the GFP specifically. Conditional over expression of Sonic Hedgehog in transgenic mice, also allowed observation over time of the effect of up regulated Sonic hedgehog signalling in normal uninjured intestinal epithelium. Changes seen in specimen treatment groups were compared using an unpaired students t-test (two groups) or one way analysis of variance (ANOVA) followed by Tukey's mutiple comparison test for mutiple groups comparisons. A probability (P) value of less than 0.05 was considered significant.?Results?600mg/kg of 5-FU injection was led to villus shortening, apoptosis and reduction of mitosis in tissues obtained 2 days after the treatment. Epithelial repair through proliferation and mitosis was demonstrated 5 days after injection, whilst tissues collected at day 9 was characterised by less extensive but ongoing repair. These time points demonstrate the reproducible injury and repair process after 5-FU treatment. An accompanying biphasic change of decrease of Hedgehog expression on day 2 and increase of Hedgehog expression from day 5 to day 9 post-injection was demonstrated through immunohistochemistry, in situ hybridization (ISH) and real time PCR. The Hh signalling target genes showed similar biphasic pattern of expression, coinciding with damage and repair phases of epithelium.Tissues obtained at day 5, after combining 5-FU with the hedgehog signalling inhibitor, showed significantly increased in apoptosis and decrease in mitosis. A significantly decreased paneth cell number among crypts cells was also demonstrated after this treatment. A prolonged phase of acute intestine inflammation in mice treated with 5-FU and inhibitor was also a feature.Dual immunofluorescence staining of A33 and GFP on Lgr5-GFP mice intestine proved that the intestinal crypt basal stem cells expressed A33. Preliminary results suggest that up-regulation of Sonic hedgehog causes increase of mitosis and hyperplasia of Paneth cell and goblet cell in the undamaged intestinal crypt after three weeks of activation.?Conclusion?5-FU induced injury of the small intestinal epithelium is a reproducible model of gut regeneration.After injury Hh expression shows a biphasic pattern, i.e. down-regulated during injury phase and up-regulated during repair phase.Rather than regulating cell proliferation, Hedgehog signalling appears to be involved in cell mitosis and differentiation within injured small intestinal crypts during repair.Hedgehog is an anti-inflammatory factor. Hedgehog plays an important role in the repair of small intestinal epithelium after acute injury...