胰岛素缓解脓毒症相关小肠上皮功能障碍涉及解偶联蛋白-2表达下调

发布时间：2018-03-12 15:20

本文选题：胰岛素　切入点：脓毒症　出处：《南方医科大学》2017年博士论文　论文类型：学位论文

【摘要】：研究背景脓毒症是高发病率和高致死率疾病。胃肠道一方面是脓毒症最常见的受累器官,另一方面,胃肠功能障碍本身可引起或者加重脓毒症,因肠道屏障受损,肠道内细菌等物质可跨过黏膜屏障进入机体循环,诱导多脏器功能障碍。极化液具有抗炎等作用,主要活性成份为胰岛素。解偶联蛋白2(UCP2)在胃肠道广泛表达,最新的研究表明,它在脓毒症时表达上调,可增加游离脂肪酸的合成,通过顺序活化蛋白激酶B(PKB)、p38丝裂原活化蛋白激酶(MAPK)、NLRP3/ASC/Caspase1信号通路,诱导促炎反应。研究目的评估胰岛素对脓毒症相关小肠上皮功能障碍的作用,了解UCP2在其中的作用。研究方法构建体内及体外两种脓毒症模型。体内模型选用SD大鼠,采用盲肠结扎穿孔(CLP)方法构建,分为空白组、假手术组、CLP处理组、CLP+极化液(GIK)治疗组、CLP+糖钾液(GK)治疗组。研究内容包括72小时大鼠死亡率、血清IL-1β/TNF-α水平、回肠组织IL-1β/TNF-α/IL-6/IL-10水平、回肠组织上皮病理HE染色结果(Chiu's评分法)、回肠组织渗透性变化(血清FD-4法)、回肠组织紧密连接蛋白ZO-1/Occludin/Claudin-1表达水平、回肠组织UCP2和NLRP3 mRNA情况、回肠组织UCP2、NLRP3和Caspase1 p10蛋白表达水平。体外模型采用人结直肠腺癌细胞株(Caco2)构建。体外细胞渗透性采用单层肠上皮细胞模型渗透性实验。采用Oug/ml、O.1ug/ml、1.0ug/ml、10ug/ml、100ug/ml不同浓度的脂多糖(LPS)溶液处理Caco2细胞,研究单层肠上皮细胞模型渗透性大小、UCP2和p-p38MAPK蛋白表达水平;100ug/mlLPS溶液预处理Caco2细胞,其后给予1.Onmol/ml、10nmol/ml、100nmol/ml的胰岛素治疗,24小时后研究单层肠上皮细胞模型渗透性大小、UCP2和p-p38 MAPK蛋白表达水平。研究结果1、胰岛素能够降低脓毒症大鼠死亡率,降低脓毒症大鼠的血清促炎因子beta IL-1 和 alphaTNF 水平;2、胰岛素能够降低脓毒症大鼠回肠组织炎症水平,缓解肠道上皮组织损害;3、胰岛素能够降低脓毒症大鼠回肠屏障功能损害,增加脓毒症大鼠回肠肠道上皮紧密连接蛋白ZO-1、Claudin-1和Occludin的表达水平;4、胰岛素可以降低UCP2和NLRP3 mRNA相对表达量,诱导UCP2蛋白、NLRP3蛋白和Caspase1 p10蛋白表达下调;5、不同浓度LPS刺激Caco2细胞,Caco2细胞单层肠上皮细胞模型渗透性呈浓度依赖性增加,UCP2和p-p38 MAPK蛋白呈浓度依赖性表达上调;6、不同浓度胰岛素刺激LPS预处理的Caco2细胞,Caco2细胞单层肠上皮细胞模型渗透性呈浓度依赖性下降,UCP2和p-p38 MAPK蛋白呈浓度依赖性表达下调。研究结论胰岛素可以降低CLP诱导的脓毒症模型全身炎症水平及死亡率,缓解回肠上皮组织炎症水平及病理损害,增加回肠上皮组织紧密连接蛋白的表达并增强回肠上皮组织屏障功能,主要机制可能涉及UCP2蛋白表达下调,并抑制下游的p38 MAPK 和 NLRP3/ASC/Caspase1 信号通路的激活。
[Abstract]:Backgroundsepsis is high morbidity and mortality of gastrointestinal diseases. On the one hand is the most common sepsis organ involved, on the other hand, gastrointestinal dysfunction itself may cause or aggravate sepsis, due to damage to the intestinal barrier, intestinal bacteria and other substances can cross the mucosal barrier into the body circulation, induced multiple organ dysfunction. The polarization liquid has anti-inflammatory effects, the main active ingredient for insulin. Uncoupling protein 2 (UCP2) is widely expressed in the gastrointestinal tract, the latest research shows that it is up-regulated in sepsis, can increase the synthesis of free fatty acids, through the sequential activation of protein kinase B (PKB), p38 mitogen activated protein kinase (MAPK), NLRP3/ASC/Caspase1 signal pathway, inducing pro-inflammatory response. Objective assessment of insulin on sepsis related intestinal epithelial function, in which UCP2 for the understanding of research methods constructed in vivo. And in vitro two sepsis model. In the model of SD rats by cecal ligation and puncture (CLP) method to construct, divided into control group, sham operation group, CLP group, CLP+ (GIK) GIK treatment group, CLP+ glucose potassium (GK) treatment group. The study includes 72 hours the rat mortality, serum IL-1 levels of /TNF- alpha beta, /TNF- alpha beta IL-1 in ileal tissue /IL-6/IL-10 levels, pathological HE staining of ileum epithelial (Chiu's score), ileum permeability (serum FD-4), ileum tight junction protein expression level of ZO-1/Occludin/Claudin-1 UCP2 and NLRP3 mRNA, ileum, ileum UCP2. The level of NLRP3 and Caspase1. The expression of P10 protein in vitro model using human colorectal adenocarcinoma cell line (Caco2) was constructed. In vitro cell permeability by intestinal epithelial cell monolayer permeability experiment. By using Oug/ml, O.1ug/ml, 1.0ug/ml, 10ug/ml, 100ug/ml The concentration of lipopolysaccharide (LPS) treated Caco2 cells, intestinal epithelial cell monolayer permeability model size, UCP2 and p-p38MAPK protein expression; pretreatment of Caco2 cells with 100ug/mlLPS solution, then given 1.Onmol/ml, 10nmol/ml, 100nmol/ml, insulin therapy, after 24 hours of intestinal epithelial cell monolayer permeability size, the level of UCP2 and p-p38 the expression of MAPK protein. Results: 1, insulin can reduce the mortality of septic rats, reduce the inflammatory factor beta IL-1 and serum alphaTNF level in rats with sepsis; 2, insulin can reduce the level of inflammation of ileal tissue in septic rats, alleviate intestinal epithelial tissue damage; 3, insulin can reduce the ileal barrier function septic rats damage, increased in sepsis rat ileum intestinal epithelial tight junction protein ZO-1, expression of Claudin-1 and Occludin; 4, insulin can reduce UCP2 and NLRP3 The relative expression of mRNA induced by UCP2 protein, expression of NLRP3 protein and Caspase1 p10 protein; 5, different concentrations of LPS stimulated Caco2 cells, Caco2 cells of intestinal epithelial cell monolayer permeability increased in a concentration dependent manner, UCP2 and p-p38 MAPK protein in a concentration dependent expression; 6, different concentrations of insulin stimulated LPS pretreatment Caco2 cells, Caco2 intestinal epithelial cell monolayer permeability showed a concentration dependent decrease of UCP2, p-p38 and MAPK protein expression in a concentration dependent manner. Conclusion insulin can reduce the whole body level of inflammation and mortality of sepsis model induced by CLP, relieve inflammation level and pathological damage of ileum epithelial tissue, increased expression of ileal epithelial tissue tight junction proteins and enhance the ileal epithelial barrier function, the main mechanism may involve the down-regulation of UCP2 protein expression and inhibit the downstream of the p38 MAPK and NLRP3/ASC/C The activation of the aspase1 signal pathway.

【学位授予单位】：南方医科大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：R459.7

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