| Objectives:To create the animal model with severe acute pancreatitis which can adapt to scientific research,and to investigate the effects of Qingyi granules on the intestinal mucosa barrier in rats with severe acute pancreatitis(SAP). Methods:Healthy male Wistar rats were divided into normal group,sham operation group and model A,B,C group,Severe acute pancreatitis model was induced by retrograde injection of sodium deoxycholate into the biliopancreatic duct in rats.,the sham operation group and model A,B,C Group rat,respectively,by retrograde injection of saline and 2%,3.5%,5%sodium deoxycholate,observed mortality and the extent of pancreatic injury;then 150 healthy male Wistar rats were randomly divided into 3 groups:sham-operation group,model group and treatment group.The model group and the treatment group were retrograde biliopancreatic injection of 3.5%sodium deoxycholate to create SAP model,and treatment group were given orally Qingyi granules at 2 hours before induction of SAP,at 6h,18h,30h and 42h after SAP,the model group were given placebo in the same way.The levels of serum amylase and diamine oxidase were determined,the histopathology of pancreatic and terminal ileum tissue were examined by hematoxylin and eosin(HE) staining at 0h,6 h,12 h,24 h and 48h after SAP and histologic assessment was performed.At the same time,we measured intestinal villus height,mucosal thickness and detection of small intestinal mucosal tight junction protein Claudin-1 expression by immunohistochemistry.Results:1.Sham-operated group was no death records,the mortality of models A,B,C groups after 72h were 20%,50%,70%;the level of amylase in model B group after 6 h up to 9023.0±1924.5 U/L and was a gradual decline over time,after 72 h reduced to 2245.3±271.6 U / L,but still higher than the sham-operated group and the normal group and were statistically significant(p <0.05);In model B group,the pancreas showed that interstitial edema,red blood cell leakage,inflammatory cell infiltration, acinar cells edema,point sheet hemorrhage,focal degeneration and necrosis. Pathology score of model B group and sham-operated group in every time point 9.89±2.11 vs 1.27±1.13,10.25±1.98 vs 1.32±1.28,11.71±2.11 vs 1.03±0.11, 13.85±1.05 vs 1.01±0.19,13.89±0.89 vs0.98±0.07 were showed difference significantly.2.Model rats showed apathetic,erect hair,lying tired paucimobilis;In treated group rats showed better mental state,activities of large increase in the number of defecation,perianal obvious pollution.Sham-operated group without death records, the mortality of rats with SAP were lower in treatment group.The mortality of rats with SAP in model group and treatment group were 0%(0/10),20%(2/10),40% (4/10),50%(5/10) and 0%(0/10),20%(2/10),30%(3/10),30%(3/10) respectively.The mortality of the treatment group in 24 h was lower than the model group(p <0.05).3.The level of amylase in sham-operated group appeared transient increase after model 6h and then returned to normal;In model group model,the level of amylase was highest after model 6 h,and gradually decreased with time,but in the 48 h was still higher than sham-operated group(p <0.05);Compared with the model group,the level of amylase in treatment group 12 h after began to reduce(p <0.05),in 48 h they showed no differences4.The level of serum DAO in sham-operated group before and after operation was no change (P> 0.05);The levels of serum DAO in treatment group after 24 h and 48 h was lower than the model group(p <0.05).5.After model 6h in sham-operated group acinar cells can be seen with mild edema, no bleeding and necrosis.In model group,the pancreas was Observated that interstitial edema,red blood cell leakage,inflammatory cell infiltration,acinar cells,edema, patchy hemorrhage point,focal degeneration and necrosis,and some glandular dilatation,the pathology score of pancreatic tissues gradually increased.The changes with 24 h and more in treatment group was decreasing,which was significance in statistics.6.Under light microscope,the villi structural integrity of the intestinal mucosa in sham-operated group was showed a high columnar epithelium,interstitial edema,no neutrophil infiltration.In model group,the structure of intestinal mucosa was damaged significantly in a progressive manner,particularly in the most obvious mold 24h,showing interstitial edema in rat ileum and a bit like bleeding,the expansion of lymphatic vessels,but was also showed that a large number of neutrophil infiltration with lymphoid follicular hyperplasia,thickening of intestinal villi edema,high degree of shortening,there was patchy epithelial necrosis of the top,falling,goblet cell decrease with disorder;mucosal thickness were becamed thinner,mucosa with sparse glands,glands reduced;When compared with the model group,the treatment of traditional Chinese medicine Qing Yi granules 12h in the model group reduced the pathological damage,manifested as reduced inflammatory cell infiltration,villous injury to reduce shedding,mucosal thickness and villous height were improved,24h after the beginning of the model with significant differences.48h after the operation, the indicators were close to normal.7.The claudin-1 of Small intestinal mucosa in each group were distributed in intestinal epithelial tight junction of cell membranes(including the cell membrane at the top),cytoplasmic area of membrane proximal appeared a small amount of staining, with staining brown granules,but nucleus and nuclear membrane without expression. The Claudin-1 protein of sham-operation group expression in the cell membrane and was strongly positive;Compared with the sham-operated group,the expression of claudin-1 was low in model group(p <0.05);Compared with the model group,the Claudin -1 of treatment group at different time points after model expression was higher(p < 0.05)Conclusion:1.Application of intravenous catheter system from duodenal catheter by retrograde cholangiopancreatography injection of 3.5%sodium taurocholate to establish SAP model(0.1ml/100g weight,speed of injection 0.15ml/min) is a improved model from preparation Aho SAP method,and is a good research animal model for SAP.2.Early application of Qingyi granules can improve the damage of pancreatic tissue in rats with SAP,reduce the intestinal mucosa and intestinal villus atrophy,improve intestinal tight junction protein Claudin-1 expression levels,reduce the permeability of intestinal mucosa,and decrease mortality.
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