【作者】 张剑彬；

【导师】 甘华；

【作者基本信息】 重庆医科大学 ， 内科学， 2010， 博士

【摘要】 虽然近年对各种原因诱发的全身炎症反应综合症(SIRS)和多脏器功能衰竭(MODS)从认识到治疗均有新的进展,但MODS仍然是非心血管ICU危重病患者的主要并发症和首要死因,肠道作为SIRS和MOF的“枢纽器官”和炎症介质的扩增器可能起了重要作用。现已证实,肠道粘膜屏障受损时,肠道内的细菌和毒素等发生移位、促炎介质经通透性增高的肠道屏障进入血液循环使机体出现内源性、自毁性失控的全身炎症反应,加重组织和器官的进一步损害,最终导致MODS的发生。肠道不仅是一个营养吸收器官而且也是有效阻挡肠腔内各种有害物质如细菌和毒素进入血液循环的屏障。肠黏膜上皮组织正常结构和功能的维持在防止肠道内大分子毒素和细菌跨上皮细胞和细胞旁路进入血液循环中起关键作用。肠黏膜上皮细胞间有一种特殊的结构叫紧密连接,由多种功能各异的蛋白质包括Occludin和ZO-1等组成并包绕在上皮细胞的腔侧端,受到细胞因子的高度调节并影响整个肠黏膜屏障的通透性。该结构可有效地阻止肠腔内细菌、毒素及炎性介质等物质的旁细胞转运,维持肠粘膜上皮屏障功能的完整,并对其所围绕的细胞造成膜质的区域性差异,使这些区域可进行专一的功能活动,如离子的定向转运等,其结构破坏可导致肠黏膜通透性增高,使得腔内大分子毒素穿过肠道正常保护层进入体内循环。研究发现在炎症性肠病和感染性小肠炎中,紧密连接结构蛋白解聚和重组是其肠道屏障功能受损的主要病理改变。一些促炎介质可下调Occludin和ZO-1的表达并使其发生重排。最近研究发现,肠上皮细胞内iNOS和NO表达增加与肠道粘膜上皮细胞受损及紧密连接结构蛋白Occludin和ZO-1的表达下降有密切关系。在病理状态下,NO主要是由iNOS大量合成。iNOS可被细菌或细胞因子诱导产生,在介导炎症因子导致的肠上皮通透性增高中起了重要作用。最近报道了炎症性肠病出现的肠黏膜屏障功能破坏与p38MAPK信号通路活化有关。p38 MAPK是一条重要的丝氨酸/苏氨酸蛋白激酶信号转导通路,与炎症、应激反应的调控有密切关系。p38 MAPK在受到紫外线照射、TNF、IL-1等刺激时通过蛋白激酶级联反应而使p38 MAPK酪氨酸发生磷酸化,p38 MAPK被磷酸化激活后进入细胞核内或转移到其他部位,发挥对应激条件下的的细胞免疫调节和炎症反应调控。MODS患者肠道上皮细胞内p38MAPK信号通路是否发生活化,是否调控iNOS的表达进而影响紧密连接结构蛋白表达和重组,目前尚未见报道。CBP已成为危重患者的治疗手段,尤其对多器官功能衰竭的抢救工作带来了极大方便和成为有效的救治措施,在MODS的治疗中CBP不仅作为一种血液滤过技术来清除炎症介质,而且在治疗中发挥了强大的免疫调节作用,这是CVVH治疗MODS新理念的一个重要依据。但是, CVVH是否通过改善肠黏膜屏障功能,减少细菌和毒素移位,减轻组织和器官损伤从而改善MODS的预后,目前未见文献报道。在本研究中,我们观察CBP对肠黏膜紧密连接结构蛋白、iNOS的表达和P38MAPK活化的影响,探讨CVVH改善MODS患者肠粘膜屏障功能障碍的机制。方法:1.收集了2007年12月至2009年5月从我院各科室病房转到中心ICU治疗的22例MODS患者作为研究对象,根据患者的预后分为存活组和死亡组。以20例健康志愿者作为正常对照。MODS的诊断依据美国胸科医师协会/危重病医学学会大会制定的标准。所有病人在确诊MODS之后的4小时之内开始在常规综合治疗基础上联合连续性静脉血液滤过(CVVH)治疗,分别于CVVH前(0h),CVVH治疗开始后6h,12h和20h时采集静脉血,无菌塑料试管盛装,在室温下2000g离心10分钟,分离血清,小量分装后于-70℃贮藏待用,同时收集治疗2hr时的置换废液。以血清二氨氧化酶(ADO)、D-乳酸和内毒素水平以及肠黏膜上皮细胞的通透性作为评价肠黏膜屏障功能的指标,以紧密连接蛋白分布和表达的变化作为肠黏膜破坏的标志;动态观察CVVH治疗前后患者血压、心率、呼吸、APACHII评分、血清二氨氧化酶(ADO)、D-乳酸和内毒素水平的变化情况。2.分光光度法检测血清二胺氧化酶和D-乳酸水平,鲎试剂偶氮基质显色法定量检测血清内毒素水平。3.用Caco-2细胞在体外建立单层肠粘膜上皮模型,采用MODS患者CVVH治疗前后各时间点无菌收集的血清和治疗2hr时的置换废液体外刺激培养,分别以健康正常人血清和置换液培养作为对照。Fluorescence reader检测单层肠粘膜上皮细胞对荧光素标记的葡聚糖4000(FD4)的通透量和millicell-ERS电阻仪检测其跨膜电阻(TER)来评价肠粘膜上皮的通透性及CVVH对其影响。4.免疫荧光染色检测上述各组刺激培养的Caco-2单层肠粘膜上皮细胞紧密连接蛋白Occludin和ZO-1的分布和重排情况;Wes- tern blot检测细胞紧密连接蛋白Occludin和ZO-1表达水平的变化。5.Western blot检测上述各组刺激培养的上皮细胞中磷酸化P38M- -APK的蛋白表达水平的变化。6.应用RT-PCR方法检测上述各组刺激培养的上皮细胞内iNOS的基因表达水平的变化。7.用比色法检测上述各组刺激培养的上皮细胞内NO水平的变化。结果:1.CBP治疗过程中MODS患者临床表现和全身状况的变化:MODS患者在CVVH治疗时血流动力学稳定,收缩压,舒张压和平均动脉压均无明显波动,心率在CVVH治疗6小时后有所下降,此后一直稳定至治疗结束。12个患者在开始行CVVH治疗时需要至少一种血管活性药物维持正常血压,其中有3个患者同时需要两种药物联合,有2个患者同时需要三种药物联合。CVVH治疗后,上述8位患者血管活性药用药剂量或数量减少,4位患者血管活性药物维持在治疗前水平。15位合并代谢性酸中毒的患者在CBP治疗12-20小时后得以纠正,10位患者的急性肾功能衰竭在CBP治疗后也得以减轻。每个患者根据病情需要进行1-5次CVVH治疗,其中12例患者存活,10例患者死亡。MODS患者死亡组的APACHEⅡ评分显著高于存活组,治疗20小时后,两组患者APACHEⅡ评分都较治疗前下降,存活组下降程度显著大于死亡组。2.MODS患者肠道屏障功能的变化及CBP对其影响:与正常对照组相比,MODS患者存在明显的肠黏膜屏障功能障碍,表现为MODS患者血清DAO、D-乳酸和内毒素水平以及MODS患者血清刺激培养的单层肠粘膜上皮细胞的通透性明显增高。与MODS存活组相比,MODS死亡组外周血DAO、D-乳酸和内毒素水平及其血清刺激培养的肠粘膜上皮细胞的通透性增高更显著。在CVVH治疗过程中,与治疗前相比存活组外周血内毒素水平在6h明显下降,死亡组在12小时下降,两组患者外周血DAO水平在20小时明显下降;存活组患者下降幅度显著高于死亡组。3.MODS患者血清诱导的单层肠粘膜上皮细胞通透性的变化及CBP治疗对其影响:与正常对照组相比,MODS存活组和死亡组患者治疗前血清诱导的单层肠黏膜上皮细胞对FD4的通透性增高和跨上皮细胞TEER下降,CBP治疗6h血清诱导的单层肠黏膜上皮细胞对FD4的通透性下降,并且TEER明显增加,12h达最佳效果, 20h时效果略有下降。尤其以存活组患者单层肠黏膜上皮细胞通透性改善更为显著,显著优于死亡组;与置换液对照组比较,置换废液诱导的单层肠黏膜上皮细胞对FD4的通透性增高和TEER下降。4. MODS患者血清诱导的单层肠粘膜上皮细胞紧密连接蛋白重排及CBP对其影响:正常单层肠粘膜上皮细胞的Occludin和ZO-1主要分布在细胞周边并在细胞与细胞接触的部位形成连续光滑的紧密连接条带,与细胞构成蜂窝状。与正常对照组相比,MODS患者血清培养的单层肠粘膜上皮细胞可见occludin和ZO-1分布发生改变,其染色减弱,连续性中断,部分断裂且细胞间连接松散,CVVH 6h时机密连接蛋白重排减轻,细胞间紧密连接不规则和松散得以改善,尤以12小时最为明显,并持续到20小时。5. MODS患者血清诱导的单层肠粘膜上皮细胞紧密连接蛋白表达变化及CBP对其影响:与正常对照组相比,CVVH治疗前MODS患者血清刺激培养的单层肠粘膜上皮细胞Occludin和ZO-1蛋白表达水平下降,治疗6小时后Occludin和ZO-1蛋白表达水平升高,12小时后表达水平升高接近正常水平,持续到治疗20小时。6. MODS患者血清诱导的单层肠上皮细胞iNOSmRNA和NO表达水平的变化及CBP对其影响:与正常对照组相比,CVVH治疗前MODS患者血清刺激培养的单层肠粘膜上皮细胞iNOS基因表达和NO生成水平明显升高,在CVVH治疗6小时后,单层肠黏膜上皮细胞iNOS基因表达和NO生成水平下降,治疗12小时达最佳效果,20小时效果略有下降,但与治疗6小时相比无统计学意义。7.MODS患者血清诱导的肠上皮细胞P38 MAPK信号通路活化与iNOSmRNA表达的关系及其对TJs蛋白表达和分布的影响:与正常对照组相比,MODS患者血清刺激培养的单层肠粘膜上皮细胞P38MAPK活性明显增高。P38MAPK信号通路抑制剂SB203580干预后,iNOSmRNA表达和NO生成水平明显降低;同时Occludin和ZO-1蛋白表达水平和组合也明显改善。8.CVVH对P38MAPK信号通路的影响:与CVVH治疗前MODS患者血清刺激培养的单层肠粘膜上皮细胞相比,在CVVH治疗6小时后的单层肠黏膜上皮细胞P38MAPK表达水平下降,治疗12小时达最佳效果,20小时效果略有下降,但与治疗6小时相比无统计学意义。结论:1.MODS患者存在肠粘膜屏障功能损害,其损害程度可能与患者的病情严重性和预后有相关。2.连续性血液净化(CBP)治疗不仅有效改善MODS患者的全身状况如APACHEII评分,同时也改善了患者肠粘膜屏障功能的损害。3. MODS患者肠粘膜上皮细胞紧密连接蛋白Occludin和ZO-1重排和表达下调参与了其肠粘膜屏障损害的过程,连续性血液净化治疗有效改善了Occludin和ZO-1连接蛋白的重排和表达。4.CBP治疗对MODS患者肠粘膜屏障的这种保护作用可能与其限制SIRS和氧化应激进而下调P38MAPK信号通路活性,抑制iNOSmRNA表达,减少NO生成有关,从而改善了MODS患者的肠粘膜屏障功能更多还原

【Abstract】 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.更多还原