The Variation Of Intestinal Microecology In Rats With Cirrhosis And Portal Hypertension And The Effects Of Lactulose On It

OBJECTIVE1. Establish a steady,credible rat model of cirrhosis portal hypertension. Study its pathological changes of live and intestine.2. To study the bacterial translocation, intestinal microecology, gut barrier and endotoxemia. Explore the effects of TNF-αon the Occludin protein and the intestinal barrier and its role in the pathogenesy of endotoxemia origin from intestine.3. To Research on the effects of microecological preparation-lactulose on conditioning the imbalance of intestinal flora of rats with portal hypertension, inhibiting bacterial translocation, attenuating endotoxemia and improving liver function, explore its role of mechanisms in treatment of portal hypertension.4. To Research on the effects of microecological preparation-lactulose on the intestinal microecology of rats with portal hypertension that have recived splenectomy and devascularization, to explore its significance in surgical treatment of portal hypertension.METHODS1. Models of portal hypertension were established as follows: 50% CC14 olive oil was administered parenterally on the back of rats at a dose of 0.4 mL/100 g body weight twice a week for consecutive 4 weeks. On the 5th week, the dose was changed to 0.5 mL/100 g and the medication was given twice a week for 4 weeks. The preparation of portal hypertension model lasted 10 weeks. The rats received upper abdominal CT, MR scanning. Observed the conditions of cirrhosis, splenomegaly, ascites ect. Study the pathological changes of live and intestine tissues by using HE staining, Masson staining and Transmission Electron Microscope, respectively. The diameter of portal vein and portal venous pressure was used to observe the change of flow dynamics.2. Ultra-structure of intertinal epithelial cells and villus pathological changes were observed under electron microscope., the bacterial translocation rate was calculated, The contents of cecum were examined for the growth of anaerobic bacteria. Randomly amplified polymorphic DNA (RAPD) technique was used for the analysis of DNA fingerprint. Blood was sampled from abdominal aorta for the measurement of liver function. Modified diazo-coupling method was employed for the detection of endotoxin. The expressions of TNF-αand Occludin were determined in the tissues of terminal ileum and colon by using immunochemistry and Western blot respectively.3. The treatment with lactulose was given to rats with portal hypertension: the rats were gavaged with 5 ml/kg lactulose twice daily when rats began to defecate loose stools, then carried out our research as step 2.4. The portal hypertension rats were given splenectomy and Periesophagogastric devascularization, then carried out our research as step 3.RESULTS1. The mortality rate of rat's model of portal hypertension is 28%. 10 weeks after the rats were subcutaneously injected of 50% CCL₄ olive oil on the back, weight loss was seen, CT revealed that the liver volume was reduced and spleen enlarged, which was accompanied with a small quantity of ascites in the perihepatic tissues. Pathological examination revealed plenty of pseudolobules containing fibrous septa and regenerative nodules of liver cells. The intra-abdominal pressure measurement showed that the pressure of the experimental group was significantly higher than that of the control group (15.76±2.68 mmHg vs. 7.62±2.02 mmHg, P<0.05) 2. The bacterial translocation rate was increased in portal hypertension group when compared with that in normal group (P<0.05). The number of lactobacillus and bacillus bifidus was declined and that of enterobacteria and enterococcus elevated (P<0.01). Compared with that in normal group, the liver function profile was significantly worsed in portal hypertension group (P<0.01). And the endotoxin level (0.44±0.07Eu/ml) was much higher in portal hypertension group than normals (0.20±0.08Eu/ml) (P<0.01). There were significant differences in the expressions of TNF-αand Occludin between the two groups (P<0.01).3. After treated with lactulose, the bacterial translocation rate was increased in portal hypertension treatment control group (group A) when compared with that in lactulose group (B) and normal control group (C) (P<0.05). The number of lactobacillus and bacillus bifidus was declined and that of enterobacteria and enterococcus elevated in group A as compared with those in groups B and C (P<0.01). Less bands and abnormal bands were found on the DNA fingerprint map of group A. Gene bands of dominant bacterial flora in group B were akin to those in group C. Compared with that in groups B and C, the liver function profile was significantly worsed in group A. And the endotoxin level was much higher in group A than in groups B and C. There were significant differences in the expressions of TNF-αand Occludin between group A and groups B, C (P<0.01).4. The portal hypertension rats that had received "splenectomy plus pericardial devascularization" were gavaged with lactulose. The bacterial translocation rate was increased in treatment control group postoperation (group PC) when compared with that in lactulose group postoperation (group PB) and sham operation group (group PD) (P<0.05). The number of lactobacillus and bacillus bifidus was elevated and that of enterobacteria and enterococcus declined in groups PB and PD as compared with those in group PC (P<0.01). Less bands were found on the DNA fingerprint map of group PC. Compared with that in groups PB and PD, the liver function profile was significantly worsed in group PC. And the endotoxin level was much higher in group PC than in groups PB and PD. There were significant differences in the expressions of TNF-αand Occludin between group PC and groups PB, PD (P<0.01). CONCLUSIONThe model of portal hypertension establish by subcutaneous injection of 50% CCL₄ Olive oil on the back of rats has a similar histology change to human cirrhosis. The portal venous pressure raised significantly and certain level's of port-body branches' shunts occurred, It is a appropriate model to study portal hypertension.The development of portal hypertension is accompanied by the damage of intestinal barrier. The raise of intestinal permeability is the main reason for intestinal endotoxemia. The disturbanc of intestinal microecology, bacterial translocation, dysbacteria and intestinal endotoximia occurred, meanwhile liver function got worse.The treatment with lactulose can successfully improve the intestinal environment, enhance the ability of intestine to ward off microbial colonization, reduce the bacterial translocation, and attenuate endotoxemia, thereby improving the liver function. It's these kinds of functions refer to not only portal hypertension rats that have not received operation but also those that have undergone the operation. This also provided a theoretical basis and accumulated experience for improving surgical treatment and quality of life in patients with portal hypertension and alleviating the short-term and long-term complications in clinic.