| Background and objective:Despite the recent advance in the understanding and management of systemic inflammatory response syndrome (SIRS) and multiple organ dysfunction syndrome (MODS), MODS remains the main complication and leading cause of death in non-coronary intensive care units(ICU), The intestine plays an important role as the"hinge"organ of SIRS and MODS and the amplifier of inflammatory mediators. When gut barrier is injured , intraintestinal bacteria and toxin translocate across the permeable intestinal mucosa into the peritoneal vein ,And inflammatory mediators generated in the intestine invade the intestinal blood circulation system, thereby producing an endogenous, self-destructive, and uncontrolled systemic inflammatory response in the body and increased tissue and organ injury, which eventually leads to MODS.Intestinal tract is not only to function as a nutrition absorbtion organ but also as a highly selectively permeability barrier that prevents the passage of harmful substance such as bacterium and toxins into systemic circulation.The maintenance of normal gut mucosal structure and function is essential to prevent luminal bacteria and endotoxin that nomally inhabits the gut from translocating across the intestinal mucosa into the peritoneal vein. The intact epithelium forms a relatively impermeable barrier between the lumen and the submucosa. This barrier function is maintained by a complex of proteins comprising the tight junction that is located at the sub-apical aspect of the lateral membranes. Consisting mainly of the structural proteins occludin and zonula occludens-1 (ZO-1), the highly regulated tight junction determines the overall permeability of the intestinal epithelium. The tight junction structure is fundamental to form a integrity mucosa barrier, which cause membranous regional differences around Cells their surrounding so these regions can be specific features activities such as ion transport orientation, etc. It can also effectively prevent luminal products as bacteria , toxin and proinflammatory cytokines from transferring into the systemic circulation, Disruption of the tight junction can increase the intestinal mucosal permeability, allowing luminal products to accedd normally protected layers of the intestine and penetrate into intestinal circulation. it has been reported that disruption of the intestinal epithelial barrier due to disorganization of the tight junction leads to an increase in paracellular permeability in inflammatory bowl disease and intestinal infections. Recently, upregulation of inducible NOS expression and increased NO production has been implicated as factors contributing to the decrease of tight junction protein levels. Under pathophysiologic conditions, NO is synthesized in large amounts by the inducible NO synthase(iNOS).The transcription of the gene encoding iNOS can be induced by bacteria or by proinflammatory cytokines, Which plays critical role in mediating Cytokine-induced intestinal epithelial hyperpermeability。Due to the important position of the tight junction structural proteins and iNOS in the breakdown of intestinal mucosal barrier , Controling through appropriate interventions, It has very important clinical significance in prevention and treatment of gut barrier dysfunction in patients with multiple organ dysfunction syndrome.Recently, It has been reported that p38MAPK signal pathway is related with breakdown of gut barrier in inflammatory bowl disease. p38 MAPK is an important serine/threonine protein kinase signal transduction pathway which participate in regulation of inflammation and stress response. When stimulated by ultraviolet radiation, TNF-aand IL-1, tyrosine of P38MAPK was phosphorylated by a protein kinase cascade, the phosphorylation p38-MAPK was activated and then transfer into cell nucleu or other site, Which play important role in regulating cell immunity and inflammatory reaction under stress condition. whether p38MAPK signal pathway was activated in epithelial of patients with MODS and participated in regulation of iNOS expression and thus affect the expression of tight junction structure and reorganization, has not yet been reported.CBP has become a effective treatment for critical patients, especially in the rescue of multiple organ failure .It is now considered that CBP not only as a blood filtration technology to remove inflammatory mediators but also a powerful immune regulator, This is an important basis for CVVH in the treatment of MODS. However, it remains unknown whether CBP favorably influences gut barrier dysfunction , reducing translocation of bacterium and toxins and then decreasing damage of issue and organs. To investigate the mechanism of CVVH in the treatment of intestinal mucosal barrier dysfunction in patients with MODS, we further observe the effect of CBP on expression and localization of tight junction structural protein , and expression of iNOS and activation of P38MAPK in this study.Method: 1.Twenty-two patients with MODS were recruited from all wards regardless of discipline and were subsequently transferred to the general intensive care unit, from December 2007 to May 2009. MODS was diagnosed in accordance with the American College of Chest Physicians Society of Critical Care Medicine Consensus Conference definition. Treatment was initiated within 4 hours after fulfillment of MODS criteria. Each patient received the standard CBP protocol of continuous venovenous hemofiltration (CVVH) as well as standard intensive care unit resuscitative measures including ventilation, adequate hydration, and inotropic support. Twenty healthy volunteers matched for age and sex served as control subjects. Venous blood samples were collected at 0 (just before therapy was initiated), 6, 12, and 20 hours during CVVH, and waste replacement fluid at 2 hour during CVVH was also collected, All the sample were placed in sterilized plastic tubes. Serum was separated by centrifugation at room temperature (2,000g;10 minutes), and aliquots were frozen at -70°C for subsequent procedures. The epithelial monolayer permeability,TER, and serum levels of DAO and endotoxin were used to assess epithelial function in patients who underwent CBP. Dynamic change of blood pressure, heart rate, breathing and APACHII score ,serum levels of diamine oxidase (ADO), D-lactate and endotoxin were observed during CVVH therapy in this study .2.Serum levels of DAO and D-lactate was determined by spectrophotography, and serum endotoxin level was determined by quantitative colorimetriclimulus test.3.The established intestinal epithelial monolayer model was used to evaluate changes in the permeability and resistance of epithelial cells in response to patients'sera before and during CBP treatment.4.Immunofluorescence staining of tight junction proteins occludin and ZO-1 localization in Cells treated with serum from each experience group, the expression levels of occludin and ZO-1 were also detected by Werstern blot.5. The protein expression of phosphorylation P38MAPK in Cells treated with serum from each experience group were detected by western blot.6. The mRNA expression of iNOS in Cells treated with serum from each experience group were surveyed by using RT-PCR.7.The production of NO in in Cells treated with serum from each experience group were measured by colourimetry.Result:1.The clinical manifestation and general condition: During CVVH therapy, patients exhibited stable hemodynamics.No significant drop in systolic or diastolic blood pressure, or mean arterial pressure was observed.The pulse pressure and heart rate fluctuated throughout the first 6 hours after initiation of therapy, but no significant differences were observed. When CVVH therapy began, 12 patients required inotropic support including noradrenaline, dopamine, or dobutamine; 3 of these patients required the combination of 2 drugs, and 2 patients required the combination of 3 drugs. After CVVH therapy, medication dosage was reduced in 8 patients, and remained unchanged for 4 patients. In 15 patients, acidosis was reduced, and 10 patients experienced alleviation of acute renal failure. Among MODS patients,patients who later died had significantly higher APACHEII scores compared with those who survived. APACHEII scores of patients in both groups decreased after 20hours of CVVH therapy, but the decrease was more pronouncedin surviving patients).2. Gut barrier function in patients with MODS and its change after CVVH treatment:Gut barrier dysfunction was evident in patients with MODS but not in normal controls. Gut barrier dysfunction was detected as elevated serum DAO,D-lactate and endotoxin levels in MODS patients, and increased permeability in the monolayer intestinal epithelium model exposure to serum serum from patients. Changes in MODS patients who later died (non-survivors) were more pronounced than those of survivors. After 20 hours of CVVH therapy, peripheral blood DAO levels in patients of both groups were significantly decreased compared to pre-therapy levels. Peripheral blood endotoxin levels in survivor group decreased at 6 hours while peripheral blood endotoxin levels decrease in non-survivor group at 12 hours during CVVH.; these conditions were maintained during the rest of the therapy. Furthermore, the decrease of serum endotoxin and DAO in group of survivors was more significant than the group of non-survivors.3.The change of permeability in intestinal epithelium monolayer exposure to serum serum from patients with MODS during CVVH therapy: Compared to controls, increased permeability of FD4 and decreased TER in the monolayer intestinal epithelium was found after exposure to serum from patients with MODS. At 6 hours following the initiation of CVVH, permeability of the intestinal epithelial monolayer decreased while TER of the intestinal epithelial monolayer increased in both groups; these changes were most prominent after 12 hours and were maintained for the rest of the therapy. Furthermore, the improvement of permeability in group of survivors was more significant than the group of non-survivors.4. The change of Cellular localization of occludin and ZO-1 in the intestinal epithelial monolayer exposure to serum serum from patients with MODS during CVVH therapy: Immunofluorescent staining revealed that occludin and OZ-1 were mainly distributed around the cells of the healthy intestinal epithelium and constructed smooth and continuous tight junction belts in areas of cell-to-cell contact . Changes in distribution of occludin and ZO-1 were seen in the intestinal epithelial monolayer of MODS patients, shown as weaker and interrupted staining, and ruptured and loosened junctions in some cells. After 6 hours of CVVH therapy, destruction and breakdown of occludin and ZO-1 were attenuated, and irregular and loosened tight junctions between cells were also improved; these changes were most prominent after 12 hours and were maintained for the rest of the therapy.5. The change of expression levels of Occludin and ZO-1 in intestinal epithelial monolayer exposure to serum serum from patients with MODS during CVVH therapy: Compared to the healthy control group, decreases in occluding and ZO-1 expression levels were found in MODS patients prior to treatment. The tight junction protein expression levels increased significantly after 6 hours of therapy. Expression was restored almost to normal levels after 12 hours; this increase was maintained for the rest of the therapy6.Alterations of iNOS expression and intracellular NO in the intestinal epithelial monolayer exposure to serum serum from patients with MODS during CVVH therapy: iNOS expression and NO levels were significantly elevated in the intestinal epithelium monolayer treated with serum collected from the patients before therapy, compared to cells treated with serum of healthy controls. Cells treated with serum collected from patients at 6 hours following the initiation of CVVH therapy produced significantly lower iNOS expression and NO levels, and the decrease was maintained during the rest of the therapy.7. P38 MAPK signaling pathway activation induced by serum from patients with MODS and its effect on iNOS expression and breakdown of TJs protein: Expression levels of P-P38MAPK were significantly elevated in the intestinal epithelium monolayer treated with serum collected from the patients before therapy, compared to cells treated with serum of healthy controls. After interfere with SB 203580, a inhibitor of P38 MAPK signaling pathway, iNOS expression and NO levels were significantly decreased; at the same time, the increased expression levels and improved disorganization of occluding and ZO-1 were also found.8. Alterations of P38 MAPK signaling pathway in intestinal epithelial monolayer exposure to serum serum from patients with MODS during CVVH therapy: Expression levels of phosphorylation P38 MAPK in the intestinal epithelium monolayer treated with serum collected from the patients at 6 hours during CVVH decreased significantly when compared to cells treated with serum from patients before CVVH. Cells treated with serum collected from patients at 6 hours following the initiation of CVVH therapy produced significantly lower iNOS expression and NO levels, and the decrease was maintained during the rest of the therapy, these changes were most prominent after 12 hours and were maintained for the rest of the therapy.Conlusion:1.Breakdown of gut barrier is present in patients with MODS , The severity of disease may be correlated with the degree of gut barrier injury.2. CBP therapy can not only improve general conditions, as measured by the APACHE II score, but can also effectively improve gut barrier dysfunction in patients with MODS, regardless of disease severity.3. The rearrangement and decreased expression of TJs protein Occludin and ZO-1 involved in the breakdown of gut barrier in patients with MODS, CBP can effectively attenuate intestinal hyperpermeability by decreasing the breakdown and reorganization of these tight junction proteins.4. These beneficial effects of CBP on breakdown of TJs protein in MODS patients are associated with its potential ability to limit the SIRS response and oxidative stress then inhibit P38MAPK signaling pathway activation, downregulate intestinal iNOS mRNA expression and reduce NO production.
|
PDF Full Text Request