Intestinal Tight Junction Injury And Its Relationship With The Expression Of Soluble N-ethylmaleimide-sensitive Factor Attachment Protein α (αSNAP) In Acute Pancreatitis

Background and objective:Acute pancreatitis is the clinical common acute abdominal disease, SAP patients havesevere pancreatic local damage, at the same time, often with multiple organ dysfunctionsyndrome (MODS), such as lung, liver, heart and intestines. The intestine is a major organmost susceptible to injury caused by acute pancreatitis, and also is the starter of MODSoccurred in the process of acute pancreatitis. At present, studies have been extensiveconcerned that intestinal barrier injury plays a key role in the pathophysiology of SAP.Intestinal mucosal barrier include mechanical barrier, immune barrier, chemical barrier andbiological barriers. The expression of occludin, one of the main components of the intestinalbarrier proteins, is regulated by various factors that is involved in the regulation of intestinalbarrier formation and remodeling process in severe acute pancreatitis. The αSNAP, as anovel membrane fusion protein, is abundant in intestinal epithelial cells，and involved in theregulation of intestinal barrier formation and remodeling process. This study aimed toinvestigate the role αSNAP in acute pancreatitis, its relationship between occludin andαSNAP.Methods:1. Animal experimentsMild and severe acute pancreatitis rat models were established by retrograde injectionof0.5%and3.8%sodium taurocholate solution into rat pancreaticobiliary duct respectively.The animals were killed at1,2, and3days after the injection for pathological evaluation ofpancreas and intestinal mucosa by hematoxylin and eosin (HE), levels of serum amylasewas determined by enzymatic analysis, levels of serum TNF-α and endotoxins weredetermined by ELISA kits, changes in intestinal permeability were assessed byFITC-dextran, and expression of occludin and αSNAP were assessed by western blot and immunofluorescence.2. Experiments in vitroEpithelial IEC-6cells were transfected with αSNAP shRNA lentiviruses; the apoptosiswas determined with flow cytometry (FCM), andexpression of occludin were detected byWestern blot and immunofluorescent staining.Results:1. Animal experiment1) The SAP group showed a significantly increased levels of serum amylase, TNF-αand endotoxin. At1,2, and3days, the levels of serum amylase were as follows:1243.75±157.88,1182.5±153.09,1008.13±73.29in SO;1662.5±104.68,2326.25±300.03,2325±200.48in MAP;7897.5±1006.29,5947.5±554.69,5104.38±1256.31in SAP. At1,2,and3days, the levels of serum TNF-α were as follows:11.59±5.55,17.64±5.75,14.11±4.83in SO;55.43±9.62,86.77±22.31,38.26±10.93in MAP;174.76±35.50,203.96±60.54,119.02±29.20in SAP. At1,2, and3days, the levels of serum endotoxins were as follows:9.53±7.92,8.87±4.05,8.65±5.03in SO;54.36±11.43,67.98±8.86,88.97±9.06in MAP;208.15±24.47,197.51±22.99,137.18±31.70in SAP (p＜0.05).2) Pathology of pancreatic tissue: the SO group has no obvious change; the MAPgroup has mild edema, a small amount of inflammatory cells infiltration; the SAP group hasnecrotic pancreatic tissue and a large of inflammatory cells infiltration. At1,2, and3days,the respective pancreatic pathological scores were as follows:1.88±0.34,2.09±0.26,2.00±0.68in SO;4.86±0.61;5.06±0.59,5.35±0.62in MAP;10.63±0.51,12.95±0.39,13.91±0.45in SAP (p＜0.05).3) Pathology of jejunal epithelial tissue: the SO group has no significant injury; theMAP group only has slight shortened villibut without other significant changes; the SAPgroup has evident edema, necrosis, lodging, atrophy and shedding in intestinal epithelialcells. At1,2, and3days, the respective jejunal epithelial tissue pathological scores were asfollows:1,1, and1in SO;1.50±0.54,1.88±0.35, and2.13±0.64in MAP; and3.38±0.52,4.13±0.84, and5.13±0.84in SAP(p＜0.05).4) The intestinal permeability in rats with acute pancreatitis increased significantly, at1,2, and3days, the respective values of FITC-dextran fluorescence were as follows: 11.08±4.22,8.31±1.74, and16.50±8.83in SO;75.39±34.40,123.50±5.09, and66.11±9.48in MAP; and379.34±25.38,586.52±57.35,489.76±105.36in SAP(p＜0.05).5) Compared with those in SO and MAP, expression of αSNAP and occludin in theintestinal epithelial cells in SAP downregulated significantly.2. Experiment in vitroThe apoptosis of IEC-6cells transfected by αSNAP shRNA lentiviruses increasedremarkably.The expression of occludin decreased significantly in IEC-6cells transfected byαSNAP shRNAlentiviruses.Conclusion:1. Permeability of intestinal barrier increased in SAP.2. Downregulation of αSNAP leads to reduced occludin expression, enhancedapoptosis in intestinal epithelial cells, and hence may increase intestinal permeability insevere acute pancreatitis.