外源性一氧化碳对脓毒症大鼠肠屏障功能障碍的影响及分子机制的研究

发布时间：2018-05-18 15:12

本文选题：一氧化碳 + 脓毒症　；参考：《东南大学》2016年博士论文

【摘要】：背景和目的:脓毒症是由感染或者可疑感染引起的全身性炎症反应综合征,常伴有氧化损伤、凝血功能障碍、组织灌注不足和严重的肠屏障功能障碍。其是创伤外科和ICU常见且严重的临床综合征,并可进一步发展为严重脓毒症、脓毒症性休克以及多器官功能障碍综合征(Multiple OrganDysfunction Syndrome,MODS),被认为是重症监护室(Intensive Care Unit,ICU)患者的首位死亡原因。随着人们对疾病的认识和治疗措施的改善,脓毒症的死亡率开始下降,但严重脓毒症和脓毒症性休克的死亡率仍然较高。因此,进一步寻找对严重脓毒症及其并发的MODS新的治疗措施,是目前临床上迫切需要解决的问题。肠道是脓毒症损伤受累最早的器官之一,也是应激中心,肠道受累导致肠黏膜屏障功能障碍,进而导致持续性的肠源性细菌和毒素进入组织和血液,诱导全身过度失控炎症反应,参与远隔器官损伤及MODS的发生和发展。已有研究证实,维持紧密连接的完整性和肠黏膜上皮细胞的正常功能已成为治疗脓毒症和MODS的关键所在。近来研究证实,一氧化碳(Carbon Monoxide,CO)作为一种细胞信使分子在生物体内具有抗炎、抗凋亡、舒张血管和抑制血小板聚集等多种生理学作用。在动物实验中,增加组织CO的含量能改善各种各样的胃肠道疾病,如糖尿病性胃轻瘫、炎症性肠病(Inflammatory Bowel Disease,IBD)、术后肠梗阻以及小肠移植。本研究通过建立盲肠结扎穿孔(Cecal Ligation And Puncture,CLP)大鼠脓毒症模型,给予外源性CO释放分子2(Carbon Monoxide-Releasing Molecule-2,CORM-2),观察脓毒症大鼠细胞因子形成、肠黏膜超微结构改变、紧密连接蛋白表达等的影响,并从细胞和分子水平探讨其可能的作用机制,为临床防治脓毒症的发生发展提供新的参考。本研究分两个部分:第一部分,外源性一氧化碳对脓毒症大鼠肠黏膜屏障损伤的保护作用;第二部分,外源性一氧化碳对脓毒症大鼠肠黏膜屏障损伤保护作用的机制研究。方法:(1).SD大鼠随机分为4组:假手术组(Sham);CLP组;CLP大鼠加CORM-2治疗组(CORM);CLP大鼠加无活性CORM-2(iCORM-2)治疗组(iCORM)。CLP24小时后收集血液和小肠组织标本。ELISA检测血清中TNF-α和IL-1β含量;光镜观察小肠组织病理形态变化;电镜观察小肠组织超微结构变化;Western blot检测小肠黏膜紧密连接蛋白(ZO-1,Claudin-1和Occludin)表达;免疫荧光观察紧密连接蛋白分布情况;FITC荧光标记法观察小肠通透性。并比较观察实验各组72小时生存率。(2).检测血清二胺氧化酶(DAO)活性、D-乳酸含量和小肠黏膜髓过氧化物酶(MPO)活性,RT-PCR检测小肠黏膜Rho激酶(ROCK)-1和ROCK-2mRNA表达,Western blot检测小肠黏膜磷酸化的肌球蛋白磷酸酶目标亚基1(p-MYPT-1)/肌球蛋白磷酸酶目标亚基1(MYPT-1)和磷酸化的肌球蛋白轻链(p-MLC)/肌球蛋白轻链(MLC)蛋白的表达。结果:(1).相对于Sham组,CLP组、CORM组和iCORM组三组大鼠血清TNF-α和IL-1β含量明显增加(P0.01);小肠病理损伤和紧密连接结构破坏显著加重,Chiu's评分增加(P0.05);肠黏膜紧密连接蛋白(ZO-1,Claudin-1和Occludin)含量下降;共聚焦显微镜观察肠黏膜膜紧密连接蛋白(ZO-1,Claudin-1和Occludin)分布减少;肠黏膜通透性增加(P0.05);生存率下降(P0.05)。而CORM组与CLP组、iCORM组比较,血清TNF-α和IL-1β含量降低(P0.01);小肠病理损伤和紧密连接结构破坏显著减轻,Chiu's评分降低(P0.05);肠黏膜紧密连接蛋白(ZO-1,Claudin-1和Occludin)含量增加;共聚焦显微镜观察肠黏膜膜紧密连接蛋白(ZO-1,Claudin-1和Occludin)分布增加;肠黏膜通透性降低(P0.05);生存率改善(P0.05)。(2).相对于Sham组,CLP组、CORM组和iCORM组三组大鼠血清DAO活性、D乳酸含量和肠黏膜MPO活性明显增加(P0.01);RT-PCR显示小肠黏膜ROCK-1和ROCK-2mRNA显著增加(P0.05);肠黏膜MYPT-1和MLC磷酸化水平明显增加(P0.05)。而CORM组与CLP组、iCORM组比较,血清DAO活性、D乳酸含量和肠黏膜MPO活性明显下降(P0.01);RT-PCR显示小肠黏膜ROCK-1显著下降(P0.05),ROCK-2 mRNA无明显变化;肠黏膜MYPT-1和MLC磷酸化水平明显下降(P0.05)。结论:(1)CORM-2可以缓解CLP诱导的脓毒症大鼠的肠道损伤,抑制炎症介质的释放,保护肠上皮细胞间紧密连接,改善肠黏膜的通透性,起到保护作用。最终显著提高大鼠的生存率。(2)CORM-2对脓毒症大鼠肠黏膜的保护作用可能与其抑制Rho/ROCK信号通路减少相关炎性基因和蛋白表达有关。
[Abstract]:Background and objective: sepsis is a systemic inflammatory response syndrome caused by infection or suspected infection, often accompanied by oxidative damage, coagulation dysfunction, insufficiency of tissue perfusion, and severe intestinal barrier dysfunction. It is a common and severe clinical syndrome in trauma surgery and ICU, and can be further developed into severe sepsis and sepsis. Shock and multiple organ dysfunction syndrome (Multiple OrganDysfunction Syndrome, MODS) are considered the leading causes of death in the Intensive Care Unit (ICU) patients. The death rate of sepsis begins to decline with the improvement of understanding and treatment of the disease, but the death of severe sepsis and septic shock. The death rate is still high. Therefore, further finding new treatment measures for severe sepsis and its concurrent MODS is an urgent problem to be solved at present. Intestinal is one of the earliest organs involved in sepsis injury, and also a stress center. Intestinal involvement leads to intestinal mucosal barrier dysfunction, which leads to persistent intestinal bacteria. And toxins enter tissue and blood, induce Overcontrol of systemic inflammatory response, participate in distant organ damage and the occurrence and development of MODS. Studies have shown that maintaining the integrity of close connections and normal function of intestinal epithelial cells has become the key to the treatment of sepsis and MODS. Recent studies have confirmed that carbon monoxide (Carbon Monoxid) E, CO), as a cell messenger, has many physiological functions, such as anti-inflammatory, anti apoptosis, vasodilatation, and inhibition of platelet aggregation in the organism. In animal experiments, increasing the content of tissue CO can improve a variety of gastrointestinal diseases, such as diabetic gastroparesis, inflammatory bowel disease (Inflammatory Bowel Disease, IBD), and postoperative intestinal tract (IBD). Obstruction and small bowel transplantation. In this study, a rat sepsis model of Cecal Ligation And Puncture (CLP) was established, and exogenous CO release molecule 2 (Carbon Monoxide-Releasing Molecule-2, CORM-2) was given. The effects of cytokine formation, ultrastructural changes of intestinal mucosa and the expression of tight connexin in the septic rats were observed. This study provides a new reference for the development of clinical prevention and treatment of sepsis. This study is divided into two parts: the first part, the protective effect of exogenous carbon monoxide on intestinal mucosal barrier damage in septic rats; the second part, exogenous carbon monoxide on the intestinal mucosal barrier of sepsis rats Methods: (1).SD rats were randomly divided into 4 groups: sham operation group (Sham), group CLP, CLP rats and CORM-2 treatment group (CORM); CLP rats and non active CORM-2 (iCORM-2) treatment group (iCORM) were used to collect blood and small intestine tissue specimens. Pathological changes; ultrastructural changes of small intestinal tissue were observed by electron microscopy; Western blot was used to detect the expression of compact connexin (ZO-1, Claudin-1 and Occludin) in small intestinal mucosa; the distribution of tight connexin was observed by immunofluorescence; the small intestinal permeability was observed by FITC fluorescence labeling, and the 72 hour survival rate was compared with that of the observed groups. (2). The detection of serum two Amines oxidase (DAO) activity, D- lactic acid content and myeloperoxidase (MPO) activity of small intestinal mucosa, RT-PCR detection of Rho kinase (ROCK) -1 and ROCK-2mRNA expression in small intestinal mucosa, Western blot detection of myosin phosphatase target subunit 1 (p-MYPT-1) / myosin phosphatase target subunit 1 (MYPT-1) and phosphorylated myocutaneous eggs The expression of white light chain (p-MLC) / myosin light chain (MLC) protein. Results: (1) compared to group Sham, the content of TNF- alpha and IL-1 beta in serum of three groups of group CLP, CORM and iCORM increased significantly (P0.01); the pathological damage and close connection of the small intestine increased significantly, the Chiu's score increased (P0.05); the intestinal mucosa tightly connexin Cludin) decreased; confocal microscopy showed a decrease in the distribution of close connexin (ZO-1, Claudin-1 and Occludin) in the intestinal mucosa; intestinal mucosal permeability increased (P0.05); the survival rate decreased (P0.05). The CORM group was compared with the CLP group, the iCORM group, and the serum TNF- A and IL-1 beta content decreased (P0.01); the damage of the small intestine and the destruction of the close connexion structure were significantly reduced. Light, Chiu's score decreased (P0.05), intestinal mucosal tight connexin (ZO-1, Claudin-1 and Occludin) increased; confocal microscopy was used to observe the distribution of tight connexin (ZO-1, Claudin-1 and Occludin) in intestinal mucosa; intestinal mucosal permeability (P0.05) and survival rate (P0.05). (P0.05). (2). Three groups of CLP, CLP, and groups Serum DAO activity, D lactic acid content and intestinal mucosa MPO activity increased significantly (P0.01), RT-PCR showed that ROCK-1 and ROCK-2mRNA in intestinal mucosa increased significantly (P0.05), MYPT-1 and MLC phosphorylation in intestinal mucosa increased significantly (P0.05). RT-PCR showed significant decrease in ROCK-1 of small intestinal mucosa (P0.05), no significant changes in ROCK-2 mRNA, and significantly decreased phosphorylation of MYPT-1 and MLC in intestinal mucosa (P0.05). Conclusion: (1) CORM-2 can alleviate intestinal damage in rats induced by CLP, inhibit the release of inflammatory mediators, protect the close connections between intestinal epithelial cells, and improve intestinal permeability, The protective effect of CORM-2 on the intestinal mucosa of rats with sepsis may be associated with the inhibition of Rho/ROCK signaling pathway related inflammatory genes and protein expression.
【学位授予单位】：东南大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R459.7

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