肠淋巴再灌注加重SMAO休克大鼠炎症反应的机制研究

发布时间：2018-07-27 16:15

【摘要】：肠缺血再灌注（ischemia/reperfusion, I/R）引起的肠系膜上动脉闭塞性（superior mesenteric artery occlusion, SMAO）休克，是临床常见的危重病理过程，常见于休克复苏、器官移植术后、严重创伤救治过程等。肠I/R引起肠道屏障功能障碍、细菌/内毒素移位和大量炎症介质释放，诱发多器官功能障碍综合征（multiple organ dysfunctionsyndrome, MODS），甚至危及患者生命。因此，探讨肠道I/R引起远隔器官损伤的机制以及寻找有效的干预措施，值得深入研究。随着对肠淋巴途径研究的深入，越来越多的结果表明，肠淋巴途径在肠I/R导致远隔器官损伤的发病学中具有重要作用。我所前期研究结果表明，肠淋巴再灌注（mesenteric lymph reperfusion, MLR）可加重SMAO休克大鼠肺、肾、心、肝等器官的组织学损伤，其作用机制与加剧自由基损伤、炎症损伤有关。为了进一步探讨MLR加重SMAO休克大鼠炎症反应的作用机制，本研究以中性粒细胞（polymerphonuclear neutrophil, PMN）、高迁移率族蛋白-1（highmobility group protein box-1, HMGB1）、晚期糖基化产物受体（receptorof advanced glycation end-products, RAGE）、核因子-κB（nuclearfactor-kappa B, NF-κB）为切入点，探讨启动炎症反应的多种因素在MLR加重SMAO休克器官损伤中的作用机制，为SMAO休克器官损伤的防治提供实验依据。 24只Wistar雄性大鼠，随机分为4组：SMAO组，夹闭肠系膜上动脉（superior mesenteric artery, SMA）1h，再灌注2h；MLR组，，夹闭肠系膜淋巴管（mesenteric lymphatic duct, MLD）1h，再灌注2h;SMAO+MLR组，同时夹闭MLD和SMA 1h，再灌注2h；假手术组（Sham组），在SMA与MLR下穿线。于再灌注2h后，选择固定位置留取心、肝、肺、肾组织，一部分用中性甲醛固定，石蜡包埋，切片后应用免疫组织化学染色方法观察各组织HMGB1、RAGE、NF-κBp65和髓过氧化物酶（myeloperoxidase, MPO）表达水平；另外一部分组织制备16.7%组织匀浆，应用酶联免疫方法检测RAGE、细胞间黏附分子-1（intercellular adhesion molecule-1, ICAM-1）含量。实验结果显示，MLR和sham组的各项指标未见统计学差异。SMAO组肺、肾、心、肝组织的ICAM-1含量显著高于MLR和sham组(P0.01)、MPO表达水平强于MLR和sham组(P0.05, P0.01)，SMAO+MLR组肺、肾、心、肝组织的ICAM-1含量显著高于SMAO、MLR和sham组(P0.01)、MPO表达水平在不同程度上强于SMAO、MLR和sham组(P0.05, P0.01)，结果提示MLR加重SMAO休克大鼠器官炎症反应的机制与PMN扣押于组织增多有关。SMAO组、SMAO+MLR组肺、肾、心、肝组织RAGE的含量以及HMGB1、RAGE表达均高于或强于MLR和Sham组(P0.05, P0.01)，且SMAO+MLR组的这些指标高于或强于SMAO组(P0.05, P0.01)，结果提示MLR加重SMAO休克大鼠器官炎症反应的机制与HMGB1表达增强有关。同时也发现，SMAO组、SMAO+MLR组肺、肾、心、肝组织的NF-κB表达均显著强于MLR和sham组(P0.05, P0.01)，且SMAO+MLR组各组织的NF-κB表达强于SMAO组(P0.05, P0.01)，结果表明，MLR加重SMAO休克大鼠器官炎症反应的机制与NF-κB表达增强有关。综上，一方面，MLR增加了SMAO休克大鼠各组织器官的ICAM-1表达、引起PMN黏附、扣押于组织中，过多的PMN活化加重了机体炎症反应；另一方面，MLR增加了SMAO休克大鼠各组织器官的HMGB1表达、RAGE含量与表达、NF-κB表达，从而加重了由HMGB1、RAGE、NF-κB导致的炎症反应；至于HMGB1、RAGE、NF-κB三者之间的关系，以及在MLR加重SMAO休克大鼠炎症反应中的相互作用，还需要在以后的研究中进一步验证。
[Abstract]:Superior mesenteric artery occlusion (SMAO) shock caused by ischemia/reperfusion (I/R) is a common critical pathological process in the clinic. It is common in shock resuscitation, organ transplantation, and severe trauma treatment. Intestinal I/R causes intestinal barrier dysfunction and bacterial / endotoxin translocation. The release of a large number of inflammatory mediators can induce multiple organ dysfunctionsyndrome (MODS) and even endanger the patient's life. Therefore, it is worthy of further study to explore the mechanism of intestinal I/R caused by the injury of distant organs and to find effective intervention measures.
With the further study of the intestinal lymphatic pathway, more and more results show that the intestinal lymphatic pathway plays an important role in the pathogenesis of I/R induced distant organ damage. The results of my previous study showed that intestinal lymphatic reperfusion (mesenteric lymph reperfusion, MLR) could aggravate the histological damage of lung, kidney, heart and liver in SMAO shock rats. Injury, its mechanism is related to the aggravation of free radical damage and inflammatory injury. To further explore the mechanism of MLR aggravating the inflammatory reaction in SMAO shock rats, this study uses neutrophils (polymerphonuclear neutrophil, PMN), high mobility group protein -1 (HighMobility group protein box-1, HMGB1), and late glycosylated products receptor (recept) Orof advanced glycation end-products, RAGE), nuclear factor kappa B (nuclearfactor-kappa B, NF- kappa B) as the cut in point, to explore the mechanism of initiating inflammatory reaction in MLR aggravated SMAO shock organ damage, providing experimental basis for the prevention and treatment of shock organ damage.
24 male Wistar rats were randomly divided into 4 groups: group SMAO, superior mesenteric artery, SMA 1H, and 2H, MLR group, and then the mesenteric lymphatic vessels (mesenteric lymphatic duct), then reperfusion; After reperfusion of 2h, a fixed position was selected to leave the heart, liver, lung, and kidney tissue, part of which was fixed with neutral formaldehyde and embedded in paraffin. After the section, the expression of HMGB1, RAGE, NF- kappa Bp65 and myeloperoxidase (MPO) was observed by immunohistochemical staining. The other part of the tissue was used to prepare 16.7% tissue homogenates. Enzyme linked immunosorbent assay (ELISA) was used to detect the content of RAGE and intercellular adhesion molecule-1 (ICAM-1) in -1.
The experimental results showed that the indexes of MLR and sham group were not statistically different in group.SMAO, and the content of ICAM-1 in kidney, heart and liver tissue was significantly higher than that in group MLR and sham (P0.01). The expression level of MPO was stronger than that of MLR and sham groups (P0.05, P0.01). SMAO, MLR and sham group (P0.05, P0.01). The results suggest that MLR aggravates the mechanism of organ inflammation in SMAO shock rats and PMN seizures in group.SMAO, SMAO+MLR group lung, kidney, heart, liver tissue RAGE content and HMGB1. Higher or stronger than group SMAO (P0.05, P0.01), the results suggested that the mechanism of MLR aggravated the organ inflammatory response of SMAO shock rats was related to the enhancement of HMGB1 expression. Meanwhile, the expression of NF- kappa B in the lung, kidney, heart and liver tissues of group SMAO was significantly stronger than that in MLR and sham groups. .05 (P0.01), the results showed that MLR aggravated organ inflammatory response in SMAO shock rats and increased expression of NF- kappa B.
On the one hand, MLR increased the expression of ICAM-1 in the tissues and organs of SMAO shock rats, resulting in PMN adhesion and seizure in the tissue, and excessive PMN activation aggravated the inflammatory response of the body; on the other hand, MLR increased the HMGB1 expression in the tissues and organs of the SMAO shock rats, and the RAGE content and expression, NF- kappa B expression, thus aggravated HMGB1, HMGB1, HMGB1, kappa concerned. The associated inflammatory response; the relationship between HMGB1, RAGE, NF- kappa B three and the interaction of MLR in the inflammatory response to SMAO shock rats need to be further verified in future studies.
【学位授予单位】：河北北方学院
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R363

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