Regulation Of Postoperative Ileus By The RNA Binding Protein HuR Mediated Signal Pathway P38/MK2

Objective Postoperative ileus is a common iatrogenic complication,is caused by the mechanical manipulation of the small intestine in abdominal surgery.Inflammatory mechanisms is an important reason for the occurrence and development of postoperative ileus.P38 /MK2 is an important signal pathway which mediating cell stress and immuno-inflammatory response.Now the study verifies that Hu R(Human antigen R)via P38 /MK2 signal pathways could regulate the m RNA of various inflammatory factors after the transcription level.In this study,we will connect postoperative ileus with Hu R,to observe whether it can effectively prevent and reduce the postoperative intestinal obstruction when lentivirus mediated RNA interfere suppression of Hu R expression in mice intestinal wall,then further more to dicuss Hu R regulating affection in P38 /MK2 signal pathway.Method 1.Set up the mice model of lentivirus transfect the mice’ intestine:Male c57bl/6 mouse were randomly divided into 4 group:control group(n=5),LV-GFP1 group(n=5),LV-GFP2 group(n=5),LV-GFP3 group(n=5).Control group :mice received 1.5ml 0.9%NS intraperitoneal injection.The other three group received LV-GFP with different titer intraperitoneal injection.After 28 day regular feeding,the four group mouse were killed.Under the principles of aseptic operation,open the abdominal cavity,directly observe the condition of the lentivirus transfect the mice’ intestine under the fluorescent light.Collect several jejunum tissue samples.The tissues partial were preserved in-80 freezer preservation and the rest were fixed in Formaldehyde solution.Then we used HE staining、 immunohistochemical GFP 、GFP m RNA detection and the GFP protein detection methods to determine the optimal effect titer of lentivirus celiac transfection intestinal drops of degrees.2.Set up Postoperative ileus mice model.Experimental animals were divided into four groups,Group A(control group): no surgery operation,only open abdominal cavity,then normal saline gauze to protect small intestine;Group B(operation group):operative manipulation causes postoperative ileus;Group C(lentivirus group):intraperitoneal injected Hu R-RNAi lentivirus,normal feeding after 28 days,no surgery operation,only manipulation like group A;Group D(lentivirus +operation group): intraperitoneal injected Hu R-RNAi lentivirus,normal feeding for 28 days,then operative manipulation like group B.Liquid feed one day after all manipulation.After manipulation,intestinal tissue samples and serum specimens were collected during the respective time period for related detection.RT-PCR detect the pro-inflammatory gene expression.ELISA to detect the expression of serum inflammatory cytokines.Immunohistochemical staining to detect small bowel wall tissue inflammatory cells.HE staining to detect morphology change of the small intestine.Electromyography to detect small intestine peristalsis in vitro.Protein immunoblot technology to detect small intestine tissue protein phosphated MK2 and the total MK2,to test the expression of the cytoplasm Hu R and the total Hu R.Observe the regulated effects for inflammatory reaction in the postoperative ileus,after the suppression of Hu R.Discuss Hu R regulating affection in P38 /MK2 signal pathway.Lentivirus transfects mouse model were under the fluorescent microscope,control group without fluorescent of GFP,the other three groups mice small bowel wall tissue display fluorescent of GFP.Under naked observation LV-GFP1 group diplay weak fluorescent of GFP,LV-GFP2 and LV-GFP3 group without distinction.GFP immunohistochemistry detection showed that the control mice intestinal wall has no obvious staining,LV-GFP1 group,LV-GFP2 group and LV-GFP3 mouse small intestinal wall were stained with brown color,the color of LV-GFP1 was weak,LV-GFP2 and LV-GFP3 has no obvious difference.Mice intestinal wall HE staining,:no villus atrophy,no obvious cell necrosis,no obvious inflammation cells and fibroblasts were found in all four groups of mice intestine tissue.Use RT-PCR to detect level of GFP-m RNA,Western blot to detect GFP protein expression,control group without the GFP expression,compared with LV-GFP1 group,LV-GFP2 group and LV-GFP3 group were enhanced,but no statistical difference between LV-GFP2 group and LV-GFP3.Result The comparison of each groups:results in the number of inflammatory cells in theintestines of mouse,the expression of pro-inflammatory gene and inflammatory cytokines in serum,compared group A with group B,p 0.05,with statistical significance.Compared group A with group C,there is no statistical difference(p >0.05),results in the number of inflammatory cells in the intestines of mouse,the expression of pro-inflammatory gene and inflammatory cytokines in serum.Compared with group B,the number of inflammatory cells except Resident macrophages in the intestines of mouse,the expression of pro-inflammatory gene and inflammatory cytokines in serum were down-regulated in the group D with statistical significance(p<0.05).Compare group A with group B : to the expression of phosphated MK2,(p<0.05),with statistical significance;to the total MK2,(p>0.05),with no statistical significance;to the total expression of Hu R protein,(P>0.05)with no statistical significance;to the expression of Hu R protein in the cytoplasm,with statistical significance.Compare group A with group C:the expression of phosphated MK2,the total MK2,the expression of Hu R protein in the cytoplasm,there is no statistical difference;but to the total expression of Hu R protein,(P<0.05)with statistical significance.Compare group B with group D:to the expression of phosphated MK2,to the expression of Hu R protein in the cytoplasm,to the total expression of Hu R protein(P<0.05),with statistical difference;but to the total MK2,there is no statistical difference(P>0.05).Conclusion: 1.Intestinal wall tissue of the mouse could be transfected effectively and safely by the lentivirus,and the expression of the Hu R could be depressed,and the best effect dose is 100 ul 1.0 x 108^8GUT.2,Hu R participate in regulating the occurrence and development of the inflammatory response in postoperative ileus.3,Suppression of Hu R gene expression,can effectively inhibits the postoperative ileus inflammatory reaction and relieve the wake muscle contraction force in postoperative ileus.4,Hu R could be a potential drug target,which could be uesed to prevent and reduce the postoperative ileus.5,In the postoperative ileus Hu R may be mediated P38 /MK2 signal pathway for various inflammatory factors m RNA stability adjustment.