Study Of Gut Barrier Damage Caused By Lipopolysaccharide And Intervention By Glutamine In Baby Rats

IntroductionSevere infection is a common cause of gastrointestinal dysfunction in children's life-threatening disease.Endotoxin can induce the damage of the intestinal barrier function by the release of endogenous cytokines,inflammatory mediators,the damage of intestinal immune function,increasing intestinal cell apoptosis;The damage of the intestinal barrier function result in bacterial translocation and aggravation of endotoxemia,lead to multiple organ dysfunction(failure),even death.Therefore,the treatment of gastrointestinal dysfunction is the key on the prevention and treatment of multiple organ dysfunction.Recent studies show that the effect of glutamine is a very important role in a serious illness,gastrointestinal nutrition,immunization,Tumor therapy. Particularly in critically ill patients,stress and trauma cases,disease and trauma can lead to a state that plasma glutamine levels decreased significantly.If glutamine is supplyed timely,The rate of infection and mortality can be reduced.This study using 18-day-old rats with endotoxemia model observed pathological changes of its intestinal epithelial cells and the dynamic changes of plasma D-lactate,plasma and tissue diamine oxidase.And observed the changes of this indicators after Glutamine Intervention,to explore intestinal barrier dysfunction of rats with endotoxemia and the effect of glutamine to intestinal. Material and Methods1.MaterialThe model of endotoxemia in 18 days rats was made by intraperitoneal injection of either saline or endotoxin(5mg/kg of Escherichia coli lipopolysaccharde).The glutamine group was made by intragastric administration of glutamine(2g/kg)and intraperitoneal injection of endotoxin simultaneously.5cm intestine away from ileocecal junction 12cm were collected immediately(control only)or 1.5h,6h,24h,72h,7d after injection respectively. Small intestine's contents were cleaned up by chilled normal saline.Finally the intestine tissue were put into 10%formaldehydum polymerisatum dispensing with 0.1mol/L PBS.10cm intestine away from the ileocecal junction 1cm were collected,Small intestine's contents were cleaned up by chilled normal saline. Small intestine were bloted by filter paper,added phosphate buffer(0.1M, PH7.2),made homogenate,10000g~* 30min centrifugaed,abstractd supernatant, and preserved at -20℃.The intestinal tissue(about 1.5mm×1.5 mm size) were collected from endotoxemia 1.5h group and control group,and double fixed with 2.5%glutaraldehyde and 1%osmium acid.Blood from heart about 1 ml added heparin 0.02ml(1%heparin saline solution),centrifuged by 3200r/min, 4℃for 10 minutes,separated plasma,and preserved at -20℃.Each group have 8 rats.2.MethodsPathological changes of the small intestine villi were observed by light microscope and electron microscope.Blood plasma DAO,intestinal homogenate DAO,blood plasma lactate were detected by spectrophotometer.SPSS12.0 statistical software package was used for statistical analysis.Comparison between groups was performed using analysis of variance. Results一,Results of histological observations in endotoxemia ratsIn the control group small intestine villi are slender,developed,arranged closely and intestinal structure is clear under light microscopy.In Endotoxemia 1.5h group villus of small intestinal mucosa became edema and degeneration.In 6h group villi's edema aggravate,and inflammatory cell infiltration can be seen. In 24h group folliculus lymphaticus form in villi interstitial substance and lymphoid hyperplasia is obvious.Hyperplasia of villi can be seen in 72h group and the lymphoid tissue hyperplasia is found.Intestinal repair by itself can be seen in 7d group.Under Electron microscopy,the normal control group is not any abnormal performance.In endotoxemia 1.5h group we can see that intercellular space broaden,microvilli villous atrophy,mitochondria swell seriously and become vacuolar degeneration,endoplasmic reticulum expand,ribosomes amotio and chromatin condense around the nuclear,nucleole fragmentate and disappear,we can see apoptotic bodies.二,The changes of plasma DAO,intestinal homogenate DAO,plasma D-lactate in endotoxemia RatsIn normal control,the level of DAO(U/ml)in blood plasm is 0.824±0.167, the level of DAO(U/mgpro)in small intestine homogenate is 0.332±0.062,and D-lactate(mg/L)in blood plasm is 3.388±0.423.In the five groups of 1.5h,6h,24h,72h,7d after injecting lipopolysaccharide,the level of DAO in blood plasm is 1.142±0.303,1.377±0.272,1.729±0.245,1.209±0.222,0.666±0.190, in small intestine homogenate the level of DAO is 0.266±0.039,0.240±0.039,0.220±0.039,0.258±0.055,0.275±0.037,and D-lactate in blood plasm is 4.131±0.141,4.298±0.127,4.240±0.093,4.305±0.152,4.244±0.047. 三,The changes of plasma DAO,intestinal homogenate DAO,plasma D-lactate after using glutamineThe level of DAO in blood plasm of glutamine interference 6h,24h,72h group and 3d interference in advance group is evident lower than endotoxemia corresponding phase group,and DAO in small intestine homogenate raised obviously.The level of D-lactate in blood plasm of glutamine interference 6h,24h,72h,7d group and 3d interference in advance group is evident lower than endotoxemia corresponding phase group.Conclusion1.Endotoxemia by intraperitoneal injection of LPS can made animal appear behavior and intestinal histology abnormal.2.Intraperitoneal injection of LPS can cause intestinal tissue damage, increase of intestinal permeability and intestinal barrier dysfunction,plasma DAO and D-lactate can become indicators reflecting the intestinal barrier function.3.Glutamine intervention can reduce content of plasma DAO and D-lactic acid and increase intestinal DAO's content,and Pathology results suggest glutamine can promote repair on intestinal villi.Our study indicate glutamine have a better effect of promoting repair on intestine after injury,glutamine can reduce the permeability of the intestinal barrier and has a protective effect on intestinal barrier function.