Effect Of Adrenomedullin On Intestinal Mucosal Barrier Function In A Mouse Model Of Intestinal Ischemia/Reperfusion

BackgroundIntestinal mucosal barrier is an important part of the body’s defense system, which canresist foriegn antigens, endotoxin and intestinal bacteria into the blood circulation. Intestinalischemia/reperfusion (I/R) occurs in a number of clinically relevant pathological processesincluding small intestine transplantation, burn, hemorrhagic, septicemia etc, which results inintestinal mucosal structure impaired and barrier dysfunction, and increases the intestinalpermeability. Therefore，it very necessary to develop a new and effective way for theprevention and treatment of intestinal ischemia/reperfusion of the patient. Adrenomedullinis a vasodilator peptide, under the condition of intestinal ischemia/reperfusion,adrenomedullin protect the intestinal mucosal barrier is considered as down-regulation ofinflammatory factors. Hence, the purpose of this subject was to investigate the effect ofadrenomedullin on intestinal mucosal barrier function in a mouse model of intestinalischemia/reperfusion.MethodsFirstly，Twenty-one C57Bl/6mice were randomly divided into Sham group，I/R groupand I/R+AM group.The superior mesenteric artery (SMA) was occluded for30min usingnontraumatic vascular clamps，followed by reperfusion for6h. and the small bowel washarvested. The expressions of adrenomedullin， adrenomedullin receptor (calcitoninreceptor-like receptor，receptor activity modifying protein2and receptor activity modifyingprotein3)，ZO-1，Claudin-1were detected by Real-time PCR or Western Blotting,theactivation of p-STAT1(S727)was also assayed，Ussing chambers was used to detection ofintestinal permeability.Secondly， in a vitro model， pretreatment with AM1-52and the inhibitor ofadrenomedullin receptor (AM22-52or CGRP8-37)，intestinal epithelial cells were subjected to hypoxia. Followed by hypoxia for6h，the expression of Claudin-1were assayed by westernblotting and the morphological of tight junction protein was detected by confocalmicroscopy. And Millicell ERS was used to measure the transepithelial resistance.Thirdly， followed by reperfusion for6h. Then sections were obtained forhematoxylin-eosin (H&E) staining，to observe the morphological changes of intestinalmucosa.Results1. In the intestinal I/R model，the expression of adrenomedullin was significantlyraised，and p-STAT1(S727) signaling pathway also was activated. The expression of CRLRand RAMP2mRNA was significantly increased，but the expression of RAMP3mRNA hadno significantly change.2. Hypoxia resulted in the decrease of Claudin-1expression and the loss oftransepithelial resistance. Adrenomedullin administration significantly attenuated thedecreasing of Claudin-1expression and transepithelial resistance caused by hypoxia. At thesame time，the protective effect of AM was obviously reduced after the addition ofAM22-52，while adding CGRP8-37has little effect on adrenomedullin.3. After ischemia for30min，followed by reperfusion for6h. The expression of TJprotein and mRNA was significantly reduced，p-STAT1(S727) was abnormal activated andthe transepithelial resistance was reduced. The administration of adrenomedullinsignificantly increased the transepithelial resistance and the expression of TJ proteins，andintestinal I/R induced p-STAT1(S727) activation was significantly inhibited.4. HE staining showed intestinal I/R caused villus epithelial shedding， pretreatmentwith adrenomedullin attenuated the disruption of intestinal morphological structure causedby intestinal I/R.ConclusionAdrenomedullin could attenuate intestinal mucosal barrier dysfunction by regulatingthe expression of intestinal tight junction proteins. The inhibition of p-STAT1(S727)activation may be involved in this mechanism.