Role Of USP22 In Intestinal Repair Following Intestinal Ischemia/Reperfusion Injury

Background:Intestinal ischemia/reperfusion(I/R)injury is a common and serious pathological physiological process in clinical,it usually secondary to a variety of disease states,it would be serious when lead to a systemic inflammatory response syndrome(SIRS)and multiple organ dysfunction syndrome(MODS)and even death.The intestinal mucosal barrier is an important barrier to the body against external invasion.Once the intestinal mucosal barrier is damaged,it can easily lead to translocation of endotoxin and intestinal bacter:ium,enterogenic infection and so on,eventually leading to severe multiple organ dysfunction and failure.The dysfunction of intestinal barrier has an important influence on the occurrence,development and prognosis of critical illness.Intestinal ischemia/reperfusion injury is a common cause of intestinal mucosal barrier function injury.Therefore,to strengthen the repair of intestinal mucosa,prevent or reduce the intestinal barrier function damage,there has a very important significance for prevention and control of sepsis and MODS,but so far there is no effective treatment.Among them,the small intestine ischemia/reperfusion after regenerative repair of the recovery of intestinal mucosa barrier played a vital role.However,there has a little research about this disease.This research construct the model of intestinal ischemia-reperfusion injury and to explore the characteristics and law of regenerative and repair of small intestine and USP22 to repair the important role of small intestinal mucosa regeneration.Objective:This study was designed to observe USP22 that can influence intestinal epithelial cell proliferation and repair,further explore the protective effects of intestinal IR injury.Methods:1.Fifty Sprague-Dawley male rats were randomly divided into five groups(n=10),each consisting of seven rats as follows:(1)sham group(Control group),(2)model group 1(I/R group),(3)model group 2(I/R group),(4)model group 3(I/R group),(5)model group 4(I/R group).Among them the Control groups underwent surgical procedures that were only given appropriate separation of the superior mesenteric artery without occlusion.Other groups rats in the I/R were subjected to 1h ischemia by clamping the superior mesenteric artery followed by 3,6,12 and 24h reperfusion.The intestine tissue samples were collected for histopathology assessment,detect intestinal tissue in paraffin sections PCNA expression,while intestine homogenates were used to evaluate the level of USP22 and Cyclin D1 activity.2.Using IEC-6 cell to build intestinal hypoxia/reoxygenation(H/R)model:IEC-6 cells were placed in an incubator in hypoxia 12h(5%C02,1%02),after the end of the hypoxic put them to normal conditions,the time of reoxygenation was 3h,6h,12h and 24h.With CCK-8 assay to detect the cell proliferation activity,while USP22 and Cyclin D1 levels of the cells for each packet were detected by Western-blot method.3.To study the role of USP22 in protective effect and mechanism of intestinal ischemia/reperfusion in vitro by Intervention expression of USP22 in vitro.(1).Construction the model of H/R(hypoxia 12h,reoxygenation 6h),the target sequence(siRNA)and a negative control sequence were introduced into cells with Lipofectamine3000.(2).Construction the model of H/R(hypoxia 12h,reoxygenation 6h),the plasmid containing USP22 gene and negative control plasmid were introduced into cells with Lipofectamine3000.To assess the changes of cell activity after hypoxia reoxygenation,using flow cytometry to detect the change of cell cycle,and for USP22 activity and Cyclin D1 levels of each packet cells were detected by using Western-blot method.Results:1.Compared with the Control group,the I/R group:small intestine of rats after heat 1h ischemia,then reperfusion in 0?24h,it has experienced the period of damage and repair which there are two stages of morphological changes.At 6h reperfusion injure was the most serious,the proliferation was slowest.The HE pathological of small intestine tissue was cell edema,disordered arrangement of small intestinal villus fracture,mucous membrane and submucous interstitial hemorrhage,edema and obviously ulcer formation,and extend and change over time;the results of small intestine immunohistochemical were shown that PCNA expression in intestinal tissue,USP22 and Cyclin D1 expression trends is consistent with the trend of small intestine pathological damage in HE staining.2.Build the I/R model of IEC-6 cell in vitro,compared with the Control group,the H/R group:as the reoxygenation prolonged,the change trend of cell activity and USP22 protein levels is consistent with the experimental results in the body Intestinal IR model.3.(1).in vitro model,the cell activity which in H/R+si-USP22 than H/R group decreased significantly,USP22 and Cyclin D1 expression trend significantly reduced,By flow cytometry to detect the cell cycle were block in G1 phase..(2).in vitro model,the cell activity which in H/R + USP22 plasmid than H/R group increased significantly,USP22 and Cyclin D1 expression trend rebound significantly.Conclusion:Our findings indicated that USP22 activation is responsible for the cell cycle and relative proteins after intestinal IR.Overexpression of USP22 protects against I/R-induced injury and related complications,which may via promote the proliferation of intestinal crypt cells,and restore the injured intestinal mucosa.