The Influence Of Different Nutritional Support With Compound Lactobacilli On Intestinal Barrier Function Of Severe Acute Pancreatitis In Rats And Its Mechanism

[Objective] To establish an experimental severe acute pancreatitis (SAP) model in rats for evaluating therapeutic study;based on this SAP model, to investigate influence of different nutritional support with compound lactobacilli on intestinal barrier function of SAP in rats and its mechanism, and to investigate effect of different nutritional support with compound lactobacilli on cytokines balance of SAP in serum.[Methods] 1. Establishment of model of experimental SAP in rats: 92 SD rats were divided randomly into sham-operated group (SO) and SAP group. The SAP model in rat was established by injection of 1 ml of 3.8% sodium taurocholate beneath the pancreatic capsule. 20 rats of SO group and 20 rats of SAP group were killed at 6h, 12h, 24h and 48h after operation to determine the plasm amylase and the histopathologic changes of pancreatic. The rest 32 rats of SAP were added to calculate the mortality. 2. The influence of different nutritional support with compound lactobacilli on intestinal barrier function of severe acute pancreatitis in rats and its mechanism: 96 SD rats were divided randomly into sham-operated group with early enteral nutrition support (Sham-EEN), ENN group, EEN with compound lactobacilli (EEN+ Lac) group, sham-operated group with total parenteral nutrition support (Sham -TPN), TPN and PN with compound lactobacilli (PN+ Lac) group. Half of rats in each group were killed at 4d and 7d to determine the bacterial translocation in live and mesenteric lymph node (MLN), endotoxin and diamine oxidase( DAO) in plasm, intestinal transit index, epithelial cell apoptosis in intestinal barrier (by TUNEL), protein content in small intestinal mucosa (by BCA), the expression occludin protein(by immuneohistochemistry, IHC) and mRNA (by Real-time quantitative PCR) in small intestinal mucosa, slgA content in small intestinal mucus. 3. The rats was divided and managed with the same way of the 2 section of the research. At 4d and 7d, determine the level of TNF-a and IL-10 in serum, and observe the changes of IL-10/TNF-a ratio in different group.[Results] 1. Establishment of model of experimental SAP in rats: in SAP group, Plasm amylase increased 12 hours after injection, reached peak value at 24h, and maintained at high level at 48h. There was significant difference between model group and SO group. The HE staining results showed there was pancreatic hemorrhage and necrosis, and the impairment was progressive. The mortality rate was stable within one week. 2. The influence of different nutritional support with compound lactobacilli on intestinal barrier function of severe acute pancreatitis in rats and its mechanism: (1) the result of bacterial translocation in liver and MLN: the rate of bacterial translocation was significantly higher in SAP groups (P<0.01 vs. Sham-EEN and Sham-TPN groups);it was higher in TPN group (P<0.05 vs. EEN group);it was higher in TPN 4d group (P<0.05 vs. PN+Lac 4d group). (2) The result of endotoxin in pasm: the level of endotoxin in plasm was significantly higher in SAP groups (P<0.01 vs. Sham-EEN and Sham-TPN groups);it was higher in TPN group (P<0.05 vs. EEN and EEN+Lac group);it was higher in EEN group (P<0.05 vs. EEN+Lac group);it was significantly higher in TPN group (P<0.01 vs. PN+Lac group);and it was significantly higher in 4d group (P<0.01 vs. 7d group);(3) The change of small intestinal mechanic barrier: ?intestinal transit index: the intestinal transit index was significantly higher in SAP groups except EEN+Lac 4d and PN+ Lac 7d group (P<0.01 vs. Sham-EEN and Sham-TPN groups);it was significantly lower in TPN group (P<0.01 vs. PN+Lac group);?diamine oxidase ( DAO) in plasm: the level of DAO in plasm was significantly higher in TPN group (P<0.01 vs. Sham-EEN and Sham-TPN groups);it was significantly higher in TPN 4d group (P<0.01 vs. EEN);it was higher in EN 4d group (P<0.01 vs. EEN+Lac );and it wassignificantly higher in 4d group (P<0.01 vs. 7d group), ?intestinal epithelia AI (%): the intestinal epithelia AI was significantly higher in SAP groups (P<0.01 vs. Sham-EEN and Sham-TPN groups);it was higher in TPN group (P<0.05 vs. EEN and EEN+Lac group);it was higher in TPN group (P<0.05 vs. PN+Lac group);it was significantly higher in PN+Lac 4d group (P<0.01 vs. PN+Lac 7d group);and it was significantly higher in 4d group (P<0.01 vs. 7d group);@The relative gray value of occludin protein slices by IHC: the relative gray value of occluding was higher in SAP groups (P<0.05 vs. Sham-EEN and Sham-TPN groups);it was higher in TPN group (P<0.05 vs. EEN and EEN+Lac group);it was higher in EN 4d group (P<0.05 vs. EEN+Lac 4d group);it was higher in PN+Lac 7d group (P<0.05 vs. PN+Lac 7d group) and it was higher in EN 4d group (P<0.01 vs. EN 7d group), ?the expression of occludin protein mRNA: the expression of occludin protein mRNA was lower in SAP 4d groups (P<0.05 vs. Sham-EEN 4d and Sham-TPN 4d groups) and was lower in TPN 7d group (P<0.05 vs. Sham-TPN 7d group);it was lower in TPN group (P<0.05 vs. EEN and EEN+Lac group);it was lower in TPN 7d group (P<0.05 vs. PN+Lac group). ?There is significantly positive correlation between the occluding protein mRNA level and the reciprocal of relative gray value, r=0.9216, P<0.01.(Z)sIgA content in small intestinal mucus: the slgA content in small intestinal mucus was significantly lower in SAP groups except EEN+Lac 7d group (P<0.05 vs. Sham-EEN and Sham-TPN groups);it was lower in EEN group (P<0.05 vs. EEN+ Lac group);it was lower in TPN group (P<0.05 vs. EEN and EEN+Lac group);it was lower in PN+Lac group (P<0.05 vs. EEN+Lac group) and it was lower in SAP 4d group (P<0.05 vs. 7d group). (3)protein content in small intestinal mucosa: the protein content in small intestinal mucosa was lower in SAP groups except EEN+ Lac 7d group and PN+Lac 7d group (P<0.05 vs. Sham-EEN and Sham-TPN groups);it was lower in EEN group (P<0.05 vs. EEN+Lac group);it was lower in TPN group (P<0.05 vs. PN+Lac group);it was significantly lower in TPN group (P<0.01 vs. EEN and EEN+Lac group);it was lower in PN+Lac group (P<0.05 vs. EEN+Lac group) and it was lower in EEN+Lac 4d group (P<0.05 vs. EEN+Lac 7d group) . 3. The effect of different nutritional support with compound lactobacilli on cytokines balanceof SAP in serum. (1) the level of TNF-a in serum: the level of TNF-a in serum was significantly higher in SAP group (P<0.01 vs. Sham-EEN and Sham-TPN groups);it was lower in 4d SAP group (P<0.05 vs. 7d SAP group);it was higher in EEN 4d group (P<0.05 vs. EEN+Lac 4dgroup);it was higher in TPN group (P<0.01 vs. EEN and EEN+Lac group);it was higher in TPN group (P<0.01 vs. PN+Lac group). (2)the level of IL-10 in serum: the level of IL-10 in serum was significantly higher in SAP group (P<0.01 vs. Sham-EEN and Sham-TPN groups);it was significantly lower in 4d SAP group (P<0.01 vs. 7d SAP group);it was higher in EEN group (P<0.05 vs. EEN+Lac group);it was higher in TPN group (P<0.05 vs. EEN+Lac group);it was higher in TPN group (P<0.05 vs. PN+Lac group). (3) the ratio of IL-10/TNF-a in serum: the ratio of IL-10/TNF-a in serum was lower in SAP group (P<0.05 vs. Sham-EEN and Sham-TPN groups);it was lower in EEN 4d group (P<0.05 vs. EEN + Lac 4d group);it was lower in TPN group (P<0.05 vs. PN + Lac group).[Conclusions]1. Establishment of model of experimental SAP in ratsEstablish the rat model of SAP by injection of 1 ml of 3.8% sodium taurocholate beneath the pancreatic capsule. The mortality rate was stable within one week. And the method could establish SAP model with good reproducibility. The model resembles the natural course of human SAP and will be valuable for evaluating therapeutic study on SAP.2. The influence of different nutritional support with compound lactobacilli on intestinal barrier function of severe acute pancreatitis in rats and its mechanism(1) EEN with compound lactobacilli can significantly benefit the intestinal barrier function, decreasing the occurrence of bacterial translocation and endotoxin translocation;(2) The mechanisms of positive of EEN with compound lactobacilli on the intestmal barrier function may through improving synthesis of intestinal mucous protein, decreasing the apoptosis of the epithelial cell, improving the expression of occludin protein in intestinal epithelial tight junction, and enhancing theimmunology barrier function of intestine by improving the excretion of the slgA.(3) The mechanisms of positive of TPN with compound lactobacilli on the intestinal barrier function may through the same mechanism of EEN(4) EEN is superior in benefiting intestinal barrier function to TPN.3. The effect of different nutritional support with compound lactobacilli on cytokines balance of SAP in serum(1) EEN with compound lactobacilli can significantly benefit cytokines balance of SAP in serum.(2) TPN with compound lactobacilli can significantly benefit cytokines balance of SAP in serum.(3) EEN is superior in benefiting cytokines balance to TPN.