The Effects And Its Mechanisms Of FGFR3 In Small Intestine Of Mice After Ischemia Reperfusion Injury

Injury of intestinal mucosa barrier usually results in bacteria translocation and intestinal derived infection,which plays important role in MODS.Intestinal ischemia reperfusion injury is one of the most causes resulting in intestinal mucosa barrier damages.Accordingly,it is so important to improve repair of intestinal mucosa,prevent or diminish damages of intestinal mucosa barrier function and it plays important role in prevention and treatment of sepsis and MODS,however,there is no useful measurement yet.The recovery of intestinal barrier function after ischemia/reperfusion injury has close relation with proliferation and differentiation of intestinal epithelium and vascular endothelium,and apoptosis of intestinal epithelium is indispensable in intestinal ischemia reperfusion injury. Fibroblast growth factors(FGFs) are polypeptides play a key regulatory role in intestinal epithelial developmental and functions. Engagement of FGFs via FGF receptors activates their tyrosine kinase activity,leading to receptor autophosphorylation and to the phosphorylation of intracellular targets.Among growth factors involved in gastrointestinal tract development and homeostasis,FGFs have been shown to promote the proliferation and survival of intestinal cells.However,limited data is available on the biological activities of FGF receptors in the gastrointestinal tract.Recent research had proved that FGFR3 was involved in repair of intestinal epithelium after radiation injury. While mitogen-activated protein kinase(MAPK) is important transformer through which signaling pass from cell surface to nucleus. MAPK can regulate cell growth,development,differentiation,death and intercellular synchronization,and its subset ERK1/2 signaling transmission pathway is a common pathway of many extracellular mitosis signaling resulting in cell proliferation.Wild type mice and FGFR3+ mice were used to observe their characteristics of small intestinine in development phase and changes of FGFR3 expression.Moreover,patterns of small intestine ischemia reperfusion were applied to explore the features of intestinal mucosa after I/R,besides proliferation and apoptosis of intestinal epithelium.We also observed activity of intestinal epithelial ERK1/2 and FGFR3 expression after I/R injury,thus to determine the effect of FGFR3 in small intestine of mice after ischemia reperfusion injury and to explore the molecular mechanisms of FGFR3 promoting repairment of intestinal epithelium after I/R injury.Eventually,it might provide available evidence for new measurement to improve repair of intestinal epithelium and prevent intestinal mucosa barrier function damages.Part I The effect of FGFR3 in small intestine in development phaseMain methods:Animals and groups: Mice with Fgfr3369/+ (Mutants) and their littermate controls (C57BL/6J) (controls)1. Histologic morphology of small intestines of two groups at day1,7,14,21,28,35 and their ultrastructure changes.2. Proliferation of intestinal epithelium:intestinal epithelium cells in S phase were labelled at day 7,14,21,28,35 by BrdUrd(intraperitoneally 2 hours before being killed ,100ug/g),expression were determined by immunohistochemistry.3. Expression of FGFR3 in small intestine were determined by immunohistochemistry.4.Drew RNA of small intestine of two groups at day1,7,14,21,28,35,amplifaction by RT-PCR,then determined their levels through gel electrophoresis.Main results:1. Mutant mice had lower density and their villa were lower than the controls,but to the depth of crypt,the results were conversely.2.Cells in proliferation mainly located in intestine crypt,mutant mice had more proliferation than the controls at every time point.3. Expression of FGFR3 were detected at birth(day 1),the expression were maximal from day7 to day 21,decreasing rapidly thereafter to reach the relatively low at day35.And the expression of FGFR3 also located in intestine crypt,overlapping with that of Brdu. Part II The effects and its mechanisms of FGFR3 in small intestine of mice after ischemia reperfusion injuryMain methods:1. Animals and groups: Mice with Fgfr3369/+ (Mutants) and their littermate controls (C57BL/6J) (controls).Six to eight weeks old mice were divided into normal control group,wild I/R group and FGFR3+/- I/R group,each group has 6 mice.Absolute diet 12 hours before operation,drinking water freely.Take anesthetization by 0.6% pentobarbital sodium intraperitoneally.Superior mesenteric artery(SMA) was occluded to produce ischemia of the intestine for 1 hour followed by reperfusion to reproduce I/R injury.The mice of normal control group were killed directly while the other two groups were killed after loosening arterial clamp 1hour,3hours,6 hours and 1day,3 days. Blood were collected from orbital vein sinus, then it was centrifuged at 3000 rpm for 10 minutes and then immediately stored in a -20°C icebox to determine plasma D-lactic acid level by Thermo Labsystems Multiskan Spectrum.About 1.5 cm long proximal jejulum was fixed,embedded and sliced routely,which was prepared to examine PCNA and apoptosis.The left bowel was immediately stored at -80°C to examine expression of FGFR3 mRNA and activity of ERK1/2.Main results:1. Progressively severe mucosal injury occurred 1,3,6h after reperfusion of the ischemic intestine.At the 1,3d group,the recovery of mucosal injury were observed, The damage in small intestinal epithelium were significantly lower in fgfr3+ I/R group than wild I/R group.2.Dramatically increased epithelial apoptosis occurred 1,3,6h after reperfusion of the ischemic intestine.At the 1,3d group, attenuation of epithelial apoptosis were observed.The apoptosis in small intestinal epithelium were significantly lower in fgfr3+ I/R group than wild I/R group.3.Dramatically increased crypt epithelial proliferation occurred 1,3,6h after reperfusion of the ischemic intestine.At the 1,3d group, proliferation activity gradually decreased.The proliferation were significantly higher in fgfr3+ I/R group than wild I/R group.4. The D-lactic acid level of both two groups were remarkably higher than that of normal group at every time point,and they both reached peak value at 1h after reperfusion,then gradually decreased,but the level was significantly lower in fgfr3+ I/R group than wild I/R group at 1,3,6h after reperfusion.5. The mRNA expression level of FGFR3 in small intestine of both two groups were remarkably elevated and they both reached the first peak at 1h after reperfusion,then quickly decreased to the ebb at 3h after reperfusion.They again significantly rose to the second peak and then gradually decreased,while they remained at a high level at 3d.6. The Erk level of both two groups were at constant level,while the p-Erk level of both two groups were remarkably higher than that of normal group after injury,and then gradually decreased,the mutant decreased slowly and the wild decreased to the normal level at 1d.Major conclusions:1. FGFR3 improves formation of murine intestine crypt in development phase.2. FGFR3 prohibited the apoptosis of intestinal epithelium and improved the proliferation after intestinal ischemia-reperfusion injury.3. FGFR3 can promote small intestinal epithelial recovery,which might be acquired through activation of ERK1/2 in small intestinal epithelium.