| Objective:Sepsis is not only a great threat to human health and economy,but also one of the major diseases. The mechanism by which multiple organ damage developed has been widely investigated by many scholars on sepsis, such as:lung, heart, liver, kidney, spleen, brain, etc., but few research focus on intestinal digestive system. Intestinal tract is not only a place for food digestion and absorption, but also the reservoir of bacterium in the human body. Realizing he importance of the gastrointestinal tract in the occurrence of sepsis in recent years, small intestine, as the primary organ in sepsis, has become the new therapeutic targets. Diamine oxidase (DAO) exist in the top of mucous membrane or fluff in mammals, most of which exist in the mucous membrane of small intestine villi, few exist in endometrial fluff. As a highly active enzymes in the cell, reflecting the intestinal mucosal structure and function,it is playing the part of the ideal indicator. By measuring the activity variation of DAO in serum and intestinal tissues, the intestinal mucosal barrier function can be reflected, especially as a atraumatic method in reflecting the intestinal injury and repairment. Rhubarb has been considered to be effective in the treatment of SIRS and multiple organ dysfunction syndrome (MODS)[3]. The aims about this study is that to reveal rhubarb in sepsis rats play a role of protection mechanism、curative effect by means of determine the serum DAO level、intestinal tissue homogenate DAO level and the serum TNF alpha level in each sepsis rats. In order to guide clini-cal drug use, reduce the mortality of sepsis patients, improve the prognosis in sepsis patients, reduce the economic burden of patients who with sepsis.Methods:Experimental animal grouping:adult,84healthy SD rats, purchased from experimental animal center of HeBei medical university (certificate no.1303144), male and female half, are divided into five groups according to the random Numbers:Ⅰ. blank group6, Ⅱ. Control group6(control group), Ⅲ. Cecum ligation perforation (CLP) group24, Ⅳ. Rhubarb treatment group24,24Ⅴ ulinastatin treatment group. And group Ⅲ, Ⅳ and Ⅴ were divided into four phases by the random number table method grouping: cecum ligation perforation (CLP) after6h,12h after CLP, CLP after24h,48h after CLP,each group contains6rats.Experiment preparation:before snapping the rats were weighed and recorded according to the number of the rats before experiment.Rat cecum ligation perforation (CLP) model preparation:temperature control in the laboratory24～26℃, humidity in35～45%.Well anesthetized rats were fixed in test stage, in the supine position, the abdominal skin area was disinfected by75%alcohol, in a sterile state downward abdominal midline incision, about1.5cm, the abdominal cavity was opened, step by step in caecum and pull out of the abdominal cavity.Carefully liberate end of the cecum mesenteric (be sure to avoid bleeding, otherwise easy to shorten the recovery of rats after survival time), will be within the cecum stool gently pushed into the rectum (with a group of rats, to ensure the cecum ligation in the amount of waste residue in the basic consistent, to ensure the consistency of experimental rats with sepsis), distance ileocecus silk,1.5cm, with4avoid cecum arteries, veins and ligation in vascular arch inside (guarantee each basic consistent) rats by ligation of the length of the caecum, carefully check the ligation, maintain normal intestinal pathways. With18needles at ileocecus1cm from piercing3times, a small intestinal contents of logistics.Appendix also should be brought into the abdomen, closed abdomen one by one.Bi, subcutaneous injection of saline solution3ml/100g supplement body fluid loss. Postoperative recovery under the warm light around1.5～2h, rats regained consciousness, are free to eat drink. The way of determine the sepsis building success is that:after mental burnout appears in rats less dynamic、chills and abdominal distention、canthus secretions increase、pilomotor, anal temperature measuring<34℃,after death, after execute,there was hemorrhagic drainage in abdominal cavity,swelling in the cecum、urning blac、adhesion,jejunal bo- wel bilges gas such as performance.Specimen collection and preservation:the eye blood method was taken in pursuance of4ml, put it into the common plastic tube and let stand for about30min, for its natural coagulation, precipitation, serum centrifugal10min to3000r/min, and then take the upper serum levels of about1.5ml, moved to the Eppendorf tube,-70℃refrigerator.Open,10cm away from ileocecus intestinal tissue5cm, the longitudinal cut open, ice normal saline rinse, remove blood, intestinal content, filter paper blot, weigh and intestinal tissues,1g in10ml beaker, add0.86%saline6ml cold cut up tissue homogenate and pour into glass, with3ml ice normal saline flush residual tissue, pour slurry pipe in together, fully ground, to make10%tissue homogenate, put in4℃centrifuge, centrifugal12minutes at12000r/min, and then use pipetting device for upper and serous about1.5ml, moved to the Eppendorf tube,-70℃refrigerator.Index detection; using double antibody sandwich ELISA method, serum TNF-a, DAO and DAO content in intestinal tissue homogenate were detected in four phases in each group of rats.Statistical analysis:All data were presented as mean±SD. Statistical analysis was determined by the independent Student t test and one-way ANOVA using SPSS13.0software. AP value cut-off0.05was considered significant.Results:1The concentration of serum diamine oxidase (DAO) analysisSerum DAO content were higher in overall model group than control group (P<0.05); the serum content of DAO in the Model group after die build time variable,24h,48h group and6h,12h group had statistically significant difference (P<0.01), Began elevated in6h, continue to rise in12h, peak in24h,48h maintained at a higher level.Concentration of serum DAO in Rhubarb treatment group and the ulinastatin group6h,12h shows no statistical difference compared with model group (P>0.05); DAO concentration decreased significantly in Rhubarb treatment group and ulinastatin group24h,48h compared with model group (P<0.01). No statistical difference were found between the two treatment groups in serum diamine oxidase (DAO) concentration at each time point (P>0.05).2The intestine homogenate DAO concentration analysisThere was no statistical difference between all Groups in diamine oxidase (DAO) concentrations in intestine homogenate at each time point (P>0.05).3Serum tumor necrosis factor (TNF-a) concentrations reached the peak at6h after building (57.6+7.9),12h concentration lower (49.4+2.8), statistically significant (P<0.05),24h concentration significantly lower (23.3+3.1), statistically significant (P<0.01), and48h remain low (28.6+28.6). In rhubarb treatment group and the ulinastatin group, serum concentrations of TNF-a at each time point,6h group decreased obviously compared with model group, statistically significant(P<0.01).12h group reduced significantly, statistically significant(P<0.05),24h and48h group has no significant difference(P>0.05). Serum concentrations of TNF-a at each time point in ihubarb treatment group and ulinastatin group shows no statistical difference (P>0.05).Conclusion:1Serum levels of DAO peak at6h in the rat cecum ligation perforation model, continue to rise in12h, peak in24h, maintained at a higher level in48h.still at a higher level at12h, retreated after24h, continues to fall at48h.2Serum level of TNF-a peak at6h in the rat cecum ligation perforation model, began to fall at12h, fall markedly after24h, maintains at a low level at48h.3DAO level in Intestine homogenate in each group shows no change in the rat cecum ligation perforation model.4Rhubarb and UTI can significantly reduce DAO, serum TNF-a level in the rat cecum ligation perforation model, and reduce the occurrence of systemic inflammatory response syndrome; both drugs show similar efficacy in the treatment of sepsis. |
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