| Objective:To establish a relatively simple and rapid method for determination of putrescine and quantitative determination of putrescine content in biological samples. To abserve the effect of Changyanning for intestinal morphology and the putrescine level in rats model of simple adhesive intestinal obstruction, to explore the effect of Changyanning for the simple adhesive intestinal obstruction in rats.Methods:1.The model was established.Healthy SD rats, half male and half female, were randomly divided into four groups:normal control group(normal group), intestinal obstruction model control group(model group), cefixime positive drug control group(cefixime group), with treatment of Changyanning group(Changyanning group). Four groups of rats were fasting for 12 hours, intake water freely, 10 % hydrate anesthesia was gave to general anesthesia. The rats were fixed, hair removal, disinfection shop sterile gauze, median abdominal cut 2 cm incision, separation of jejunum, the last three groups with tooth forceps crush bowel every 5mm, returned to the abdominal cavity, normal control group(the sham operation group) operation process roughly the same at model group,just after laparotomy without clamping, sutured and disinfection. The four groups of rats on the first postoperative day in the morning were given normal saline,normal saline, and same capacity of cefixime(3.2 mg·mL-1) and Changyanning(0.1075mg·mL-1) orally, once daily for seven consecutive days.2. Determination of putrescine levels in rats intestinal tissue by HPLCBoth 15 rats in normal group and model group were sacrificed at 3 h after seventh days of administration. After the last dosage of cefixime and Changyanning 3,6, 12, 24 h,6 rats in each group were sacrificed respectively. Take the jejunum tissue samples, added 5-pyridoxal phosphate enzyme extract solution and ornithine hydrochloride to produce putrescine. Use benzoyl chloride to react with putrescine,to form pre-column derivativigation, determination of putrescine concentrations of intestinal tissue of rats by HPLC.3. The effect of Chang Yanning on adhesive intestinal obstruction in ratsTook 15 rats from each of the normal group, model group, cefminox group and Changyanning group, then in to observe the general state of the rats: laparotomy general observation, to grade the degree in accordance with the Phyllips grading standards adhesion,the jejunum tissue morphological observation under light microscope,HE staining, observed the effect of Changyanning.4. Statistical methods:The hierarchical data is analyzed by Ridit, and the variance of the quantitative data is analyzed by single factor analysis of variance and nonparametric test when the variance is uneven.Results:1. The model of adhesive intestinal obstruction was successful, and the rats in normal group, diet and defecation were normal and the weight was increased. The rats in model group had the poor appetite, no defecation, less activity, weight loss,sparse hair,visible abdominal shape, extensive adhesion of intestinal tissue after laparotomy, difficult separation and bleeding.2. To establish HPLC-Pre-Column derivation method for determination of the content of putrescine in intestinal tissue. C18 column as chromatographic column,using methanol: water(60:40) as mobile phase, UV detector wavelength was 232 nm,detection sensitivity was 0.002(AUFS). The method has the advantages of simple operation, reliable, and suitable for the determination of putrescine in biological samples.The change of putrescine concentration: The level of putrescine in Cefixime group gradually decreased after the last drug treatment. After the first 3 h, the level of putrescine decreased considerably in Changyanning group, and the putrescine level was lower than Cefixime group at 6 h, but the putrescine level had mild rebound compared to the previous individual time points, it had no significant difference in Changyanning group and cefixime group at the last time point, compared with model group, the putrescine levels in the drug treatment groups were all significantly decreased in after last treatment.3. The effect of Changyanning on the rat model of adhesive intestinal obstructionGeneral condition of the rats: Normal group, diet and defecation were normal and regular, weight gain. Model group, after the operation, rats showed lose of appetite and no defecation, spirit was dispirited, hair glory, weight loss, visible abdominal shape. Cefixime group, loss of appetite after operation and no defecation,however after gastric gavage, defecation short, small and thin after, weight increased slowly. Changyanning group, after the operation, rats showed lose of appetite and no defecation, after administration of Changyanning, diet and defecation increased gradually and weight increased slowly.Abdominal and intestinal tissue general observation: Normal group, there was no abnormal changes in intestinal tissues, no intestinal adhesion was seen. Model group,the intestinal had extensive adhesion, difficult to separate and with large amount of bleeding, most of them belonged to adhesion grade 3 and grade 4. Cefixime group,adhesion area most did not exceed 20%of the wound, most of them belonged to grade2. Changyanning group, the adhesion area was not more than 10 % of the trauma,using fingers it could be separated without bleeding.Light microscopic observation: In every group, the wall structure of jejunum in rats was clear, with normal morphology. In normal group, the intestinal villi were regular, and there was no damage, edema, and occasionally inflammatory cell infiltration. In model group, intestinal villi were shorter and irregular, the number of small intestine glands increased, and visible more pan cell, the goblet cells increased,the interstitial acute and chronic inflammatory cells infiltration, and the hyperplasia of fibrous tissue. Cefixime group, the intestinal villi were significantly thickened and partly fused, the goblet cells increased, and the cells can be seen scattered in the lymph nodes. Interstitial inflammatory cells were decreased and the fibrous tissue was obviously proliferated. Changyanning group, the intestinal villi were enlarged,became longer and partly fused. The morphology of the small intestine gland was normal, and the goblet cells were increased, the stroma cells were decreased and the fibrous tissue was obviously proliferated.Conclusion:1. Pre column derivatization-HPLC method was successful established for the determination of putrescine content in biological samples. This method is simple,accurate and reproducible. Suitable for determination of putrescine in biological samples.2. There was high level putrescine of jejunum tissue in model group rats, it reduced after using drugs,the change of putrescine level of intestinal tissue can reflect the intestinal barrier damage in a certain range.3. Changyanning can reduce the level of putrescine of jejunum tissue in intestinal obstruction rats, relieved pathological damage, and make the intestinal villus and intestinal gland tends to normal, significantly reduce the effects of intestinal adhesion, and alleviate the symptoms of intestinal obstruction.Changyanning has a certain preventive effect on the simple adhesive intestinal obstruction in rats model. |
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