Mechanisms Of Intestinal Mucosa Barrier Dysfunction Induced By Cardiopulmonary Bypass In Rats And The Protective Effects Of Glutamine

Background and objective:Cardiopulmonary bypass is a severe stress which can provoke a systemic inflammatory response syndrome (SIRS). Contact of the blood components with artificial surface of the bypass circuit, ischemia-reperfusion injury, operative trauma are mainly possible causes of SIRS. The excess inflammatory reaction may contribute to the development of postoperative complications, results in multiple organ dysfunctions, which is the leading cause of death in cardiac surgery. The protection of organs during CPB is all long important in cardiovascular surgery. Over the past years, the studies were mainly focused on the important organs such as heart, lung, liver and brain. Gut is a central organ and important target organ after surgical stress, but the research of the effect of CPB on the gut barrier function is absent and its accurate mechanism is still unknown. So it is of clinical significance for lowing the morbidity and mortality to study the mechanism of injury and investigate new strategy to protect gut barrier function during CPB.The gut barrier function is at present thought to be composed of physical, chemical, biological and immunological aspects. Intestine epithelial cell (IEC) is the central part of the barrier function. Recent studies have demonstrated that apoptosis of IEC plays an important role in maintain the homeostasis of intestinal mucosa cells. Though upregulated IEC apoptosis has been implicated in the pathophysiology of several diseases, the correlation between the gut barrier function and apoptosis of IEC is unclear during CPB. ECM is another essential part of the gut barrier, which maintains the normal structure and function of gut mucosa. Laminin and collagen â…£ are important components of basement membrane of IEC, below the basement membrane is the interstitial matrix, rich in types â…  and â…¢ collagen, as well as fibronectin and various proteoglycans. Healing and remodeling of the gut mucosa after injury is critically dependent on cell-matrix interaction. Whether or not the ECM components and activity of matrix degrading proteases includingthe matrix metalloproteinases (MMPs) changed and its contribution to the barrier dysfunction is deserved to be studied thoroughly. MMPs are a large family of proteolytic enzymes responsible for ECM degradation. MMPs and their specific physiological inhibitors, TIMPs are thought to play an essential role in tissue repair, cell death, morphogenesis and inflammatory response. Under normal condition, the system of MMPs/TIMPs maintains internal equilibrium and participates in the renewing and routine metabolism of ECM of intestine mucosa. Previous studies have shown that CPB can induce the synthesis and release of MMPs. We hypothesize that the homeostasis of MMPs/TIMPs might be shifted in gut mucosa after CPB and the imbalance should play an essential role in the pathogenesis of deregulated apoptosis of IEC and gut barrier dysfunction.CPB is an expectable stress response. Adopting measures in advance to build tolerance to perioperative damage during unstressed condition and exploit natural defense mechanism may result in a more advanced method to attenuate CPB-induced inflammation and organ imperiments. However, this novel approach through the self-protective system has not yet been applied to CPB for organ protection. Heat-shock proteins (HSPs) are self-protective proteins that maintain cell homeostasis against various forms of stress as an adaptive response. HSP70, in particular, plays a vital role in cellular protection and has been detected in various tissues subject to stress. Recent studies have demonstrated that glutamine can induce the production of HSP70 and enhance cellular survival. In addition, glutamine can also attenuate inflammation and apoptosis as a signal molecule and survival factor. Supplement of glutamine orally or via TPN can improve the intestine mucosa barrier function in patients with severe burning and trauma, but the effect and mechanism of preconditioning with glutamine on the protection of gut barrier function after CPB are still unknown.Consequently, this study based on the experimental model of CPB in rats is aimed to investigate the mechanisms of intestine mucosal barrier dysfunction induced by CPB and the protective effect and mechanism of glutamine at cellular and molecular level, and to build a new strategy and provide a new target for the protection of gut barrier function during and after CPB.Methods:1) Experimental model of CPB in rats was established via left carotid and right jugularvein cannulation for arterial perfusion and venous return respectively.2) Adult SD rats, male and weighting between 350g to 500g, were randomly divided into 2 groups according to the glutamine administration, group CPB(performing CPB routinely without preconditioning with glutamine) and group G( receiving 2ml/kg body weight Ala-Gin dipeptidal solution once a day for 3 days before CPB and priming with the same dose during CPB) . Sham-operated and normal control groups were assigned at the same time.3) Plasma concentration of TNF-a and IL-6 in plasma in rats 2h after the termination of CPB was measured by radioimmunological assay; The concentration of plasma D-lactate and LPS, activity of diamine oxidase(DAO) in plasma and gut tissue were detected using spectrophotometer respectively; Pathological and morphological changes of intestine mucosa tissue were observed by optical and transelectronic microscope; TUNEL staining for apoptosis of IEC; Immunohistochemistry, immunofluorescence and picrosirius red staining were used to analysis the changes of ECM components of gut mucosa; RT-PCR and Western blot for analysis the expression of MMP-9, TIMP-1, and HSP72 (induciable type of HSP70)at the level of mRNA and protein; Gelatin Zymography for gut mucosa MMPs gelatinlytic activity;4) Statistical analysis was performed by using software SPSS 11.0. Results:The main results in this study were as follows:1) The animal model of CPB with excellent survival was successfully established in rats by minimizing the elements of bypass circuit.2) HE staining revealed that villus edema and desquamation were relatively abundant in mucosa after CPB. There was some disruption of villus in the mucosa. Ultrastructure identified that microvillus was sparse, disarranged, and even partly absent, tight junction broadened or opened. And those in SH and N group were better than group CPB.3) The rats were in the state of systemic inflammatory response after CPB. Plasma level of TNF-a and IL-6 were significantly higher in group CPB than the control groups.4) Gut barrier dysfunction emerged in the rats undergoing CPB. The activity of DAO, the concentration of D-lactate were all increased significantly in plasma after CPB, and the activity of DAO in gut mucosa was decreased markedly in group CPB than that in other twogroups. There was endotoxemia and the plasma concentration of LPS in mesentery superior vein rose markedly.5) There was positive correlation between the proinflammatory cytokine levels and the gut barrier dysfunction.6) Compared with group SH and N, the apoptotic index in group CPB was remarkably higher and there was significantly negative correlation between AI and gut barrier, which indicated that upregulated apoptosis of IEC was the cellular base of injured gut barrier following CPB.7) The content and component of ECM in gut mucosa changed distinctly after CPB. Immunohistochemistry and RIA demonstrated that laminin and collagen IV were decreased in gut mucosa after CPB, and collagen I and III were also changed markedly. They were all correlated with gut barrier dysfunction positively and with AI negatively.8) Increased expression and activation of gut-originated MMPs was the main mechanism of the pathogenesis of inflammatory reaction, apoptosis and changes of ECM. Expression of MMP-9 was enhanced markedly at mRNA and protein levels, meanwhile no significant difference was observed in TIMP-1 expression at both levels among all groups. The gelatinlytic activity of MMP-9 and MMP-2 enhanced distinctly after CPB. These results indicated that the changes of ECM after CPB were not achieved by decreased synthesis but by increased degradation.9) There was marked positive correlation between the activity of MMP-9 and the level of TNF- a in plasma, which indicated the activation of MMP-9 might contribute to the pathogenesis and development of inflammatory response after CPB.10) Comparing with group CPB, gut barrier function was improved and inflammatory response was attenuated in group G significantly. AI was decreased and the expression of HSP72 was markedly increased in group G.Conclusions:This study based on the experimental model of CPB in rats, explored the mechanisms of the pathogenesis of gut mucosa barrier dysfunction after CPB and its contribution to systemic inflammatory response at cellular and molecular levels. The results indicated that there was intestine mucosal barrier injury following CPB, and the dysfunction play an essential role in the initiation and development of SIRS after CPB. The main mechanismswere as follows: 1) Upregulated apoptosis of IEC was one of the cellular bases of intestine injury after CPB. 2) Changes of ECM in gut mucosa caused increased apoptosis of IEC, and they both directly damaged the gut barrier. 3) Increased expression and activation of MMPs broke the homeostasis of gut-originated MMPs/TIMPs, which consequently resulted in the degeneration of ECM, apoptosis of IEC and systemic inflammatory responses, was one of the molecular mechanisms of pathogenesis of intestine mucosal barrier injury. Precondition with glutamine could improve the gut barrier dysfunction induced by CPB to some extent by inducing overexpression of stress proteins such as HSP72, inhibiting apoptosis and attenuating inflammation. To summarize the results of our studies, we presumed rationally that initiating endogenous natural defense mechanisms, downregulating the apoptosis of IEC and regulating the activation of MMPs might be a new target and strategy for the protection of gut barrier function and reducing the systemic inflammatory response during and after CPB.