The Mechanisms of Gut Barrier Injury Induced by Cardiopulmonary Bypass in Rats and the Role of Intestine Microecologic Agents
|School||China Medical University|
|Keywords||CPB Barrier function occludin， ZO-1， Tight junction DAO D-lactate proinflammatory reaction Intestinal bacterial t ranslocation Probiotics Selective digestive decontamination|
IntroductionSome studies have showed that cardiopulmonary bypass (CPB) predisposes the intestine to inadequate perfusion, hypoxic injury, and increases in gut permeability. According to our knowledge, there are no previous studies investigating whether cardiopulmonary bypass disrupts intestinal mucosal barrier by altering TJs’ formation and function.Probiotics are defined as live microorganisms that, when administered in adequate amount, confer a health benefit on the host. Selective digestive decontamination (SDD) refers to the use of antimicrobials to reduce the burden of aerobic gram-negative bacteria and/or yeast in the intestinal tract to prevent infections caused by these organisms.The present study was initiated to address two important questions in the model of cardiopulmonary bypass of rats: first, the effect CPB on the function of gut barrier and the molecular mechanisms. Second, elucidated the role of protics and SDD on the gut barrier function during CPB. Understanding the pathophysiology of barrier alterations during CPB at the molecular level will allow novel and more effective treatment approaches.Materials and MethodsMaterials1. Animals: Adult male Sprague-Dawley (SD) rats weighing 350 to 450 g were used in the present study.2. Kit and drugs: kits of LPS, TNF-a, IL-6, IL-10; Diamine oxidase, D-lactate dehydrogenase; O-dianisidine, Gadaverine Dihydrochloride and Diamine oxidase; rabbit polyclonal antioccludin or antiZO-1 antibody.3. Equipment: small animal ventilator, blood gasanalyzer, specially designed membrane oxygenator, roller pump; Space monitor; Phillips CM-10 transmission electron microscope; MetaMorph image analysis system; Olympus 1000microscropic camera.Methods1. Surgical procedure for CPBExperimental model of CPB in rats was established via left carotid and right jugular vein cannulation for arterial perfusion and venous return respectively.2. Experimental designs48 SD rats were randomly divided into four groups: Group S (n=8) served as SH operation, group C (n=24) served as CPB, group Y: CPB + probiotics, group D: CPB +SDD .The rats of group C underwent 60 minutes of CPB and were sacrificed at the end of CPB (n=8) or at 2 hours after CPB (n=8) or at 4 hours after CPB (n=8) randomly.3. Observation of parameters and methods(1) The hymodynamic changes during the period of CPB.(2) The concentration of plasma D-lactate and LPS, activity of diamine (DAO) in plasma was detected using spectrophotometer respectively.(3) Plasma concentration of TNF-a, IL-6, IL-10 and sIgA were measured by radiommunological assay.(4) Pathological and morphological changes of intestine mucosa tissue and intest- inal bacterial translocation were observed by optical and transelectronic microscope.(5) Immunohischemistry and western blot were used of Occludin and ZO-1 of gut mucosa.(6) Statistical analysis was performed by using software SPSS11.0.Results1.Gut barrier dysfunction emerged in the rats undergoing CPB. The activity of DAO and the concentration of D-lactate were all increased significantly in plasma after CPB and the most highest at 2 h after CPB (P < 0. 05). They were significantly lower in group Y or group SDD, but still higher than group SH (P < 0. 05).2.The positive rate of cultured bacterial in the blood of portal vein and other organs and the plasma concentration of LPS rose markedly after CPB (P < 0. 05), and the most highest at 4h after CPB. The rate of cultured bacterial and the concentration of LPS in group Y or group SDD were significantly lower than those in group CPB, but still higher than those in group SH (P < 0. 05).3.The concentration of sIgA was decreased significantly both in plasma and in mu- cosa after CPB and in group SDD. It was higher in group Y, but still lower than in gro-up SH(P<0.05).4.HE staining revealved that villus edema and desquamation were relatively abundant in mucosa after CPB. There was disruption of villus in the mucosa. Ultrastructure identified that microvillus was sparse, disarranged, and even partly absent, tight junction broadened or opened. And those changes in group Y or SDD were less than group CPB.5.The rats were in the state of systemic inflammatory response after CPB. Plasma levels of TNF-a, IL-6 were significantly higher in group CPB, Y or SDD and the level of IL-10 was much lower than group SH. Plasma levels of TNF-a, IL-6 in group Y or SDD were significantly lower than group CPB and the level of IL-10 was much higher.6.Evaluation of ZO-1 and occludin immunostaining and western blotAll of the epithelial cells lining villi exhibited positive immuno staining for ZO-1 and occludin in group SH. In group CPB there was loss of occludin and ZO-1 expressions in about 50% enterocytes lining villi; this effect was more profound at 2 h after CPB and loss about 80%. The 65-kDa or 220-KD bands showed a significant (P < 0.05) decrease in group CPB compared with group SH and reached the lowest values at 2 h after CPB. The expressions of occludin and ZO-1 were markrdly increased in group Y or SDD, but still lower than group SH (P < 0.05).7.There was positive correlation between the proinflammatory cytokine levels and the barrier dysfunction.8.There was significantly negative correlation between occludin or ZO-1 expressions and DAO or D-lactate levels.9.There was significantly negative correlation between occludin or ZO-1 expressions and plasma TNF-a or IL-6 levels. Conclusions1. The intestine mucosal barrier was injuried following CPB. The main mechanisms were as follows: 1) proinflammatory cytokine levels increased; 2) markedly down regulates the expressions of occludin and ZO-1 proteins and sIgA in intestinal mucosa.2. Precondiction with probiotics could improve the gut barrier dysfunction induced by CPB to some extent by: 1)Inhibiting microbial pathogens growth; 2) Increasing epit-hetlial tight junction and modifying intestinal permeability; 3) Modulating immune response of intestinal epithelia and mucosal immune cells; 4) Secreting antimicrobial products;5) Decomposing luminal pathogenic antigens.3. SDD could protect intestinal mucosa to some extent by: 1) decreased the incidence of intestinal bacterial translocation occurred during CPB, so as to decrease the incidence of postoperative endotoxemia and proinflammatory reaction; 2) maybe up-regulated the expressions of occludin and ZO-1 proteins in intestinal mucosa of rats after CPB.4. Precondiction with probiotics may be more safety and economic with less side effects.