Ethanol increases post-burn intestinal permeability: IL-6-induced MLCK activation

乙醇增加烧伤后肠道通透性：IL-6 诱导 MLCK 激活

• 批准号: 8320770
• 负责人: Anita Zahs
• 金额: $ 1.06万
• 依托单位: LOYOLA UNIVERSITY CHICAGO
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-09-01 至 2012-10-12
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8320770
• 关键词: Accounting; Acute; Acute Lung Injury; Admission activity; Affect; Architecture; Bacteria; Bacterial Translocation; Blood Circulation; Blood alcohol level measurement; Burn injury; Cause of Death; Cells; Cessation of life; Chronic; Digestion; Disease; Edema; Enzymes; Epithelial; Ethanol; Functional disorder; Heart Diseases; Hospitalization; Hospitals; Immune; Individual; Infection; Inflammation; Inflammatory; Inflammatory Bowel Diseases; Inflammatory Response; Injury; Interleukin-6; Intestines; Knock-out; Knockout Mice; Knowledge; Laboratories; Lamina Propria; Lead; Life; Lung; Lymphatic; Lymphatic System; Maintenance; Malignant Neoplasms; Mediating; Morbidity - disease rate; Movement; Mucosal Immunity; Mus; Myosin Light Chain Kinase; Organ; Patients; Permeability; Pneumonia; Predisposition; Proteins; Rho-associated kinase; Role; Sepsis; Serum; Signal Transduction; Staining method; Stains; Testing; Tight Junctions; Time; Tissues; Tumor Necrosis Factor-alpha; United States; Up-Regulation; Villus; Western Blotting; Wild Type Mouse; abstracting; alcohol exposure; binge drinker; commensal microbes; cost; cytokine; gastrointestinal; ileum; immunosuppressed; injured; mortality; myosin phosphatase; neutralizing antibody; novel therapeutics; prevent; problem drinker; response; src-Family Kinases; vascular bed

6. Abstract Ethanol is a common factor in traumatic injury, including burn injury. Previous studies from our laboratory indicate that ethanol increases both pulmonary and gastrointestinal inflammation as well as susceptibility to infection in burn-injured mice; however, the mechanisms of these responses are not entirely known. Moreover, after burn injury, bacteria and their products leak out of the intestinal lumen and into the bloodstream. These bacteria can disperse throughout the body leading to pulmonary damage, acute lung injury, sepsis, and death. Ethanol in combination with burn injury has been shown to decrease intestinal barrier function greater than either insult alone. Long (210 kDa) smooth muscle myosin light chain kinase (MLCK), an enzyme important for epithelial tight junction maintenance, has been implicated in barrier alterations after burn injury or ethanol exposure alone. With this knowledge, we hypothesize that the combination of acute ethanol exposure and burn injury causes an increase in intestinal barrier dysfunction due to interleukin-6-mediated activation of myosin light chain kinase. To test this hypothesis, three aims are proposed: to determine whether 1) elevated myosin light chain kinase activation mediates the intestinal permeability alterations in mice receiving acute ethanol and burn injury treatment, 2) interleukin-6 (IL-6) activates myosin light chain kinase and, 3) IL-6 mediates MLCK activation after ethanol exposure and burn injury. Movement of a fluorescent- tagged protein out of the ileum lumen to the bloodstream along with immunofluorescent staining for tight junction protein localization will be performed to investigate intestinal barrier function and integrity. MLCK activation will be determined by Western blot analysis. Inhibition and knock out of MLCK will be employed as well to examine the role of this molecule on intestinal permeability after acute ethanol and burn injury exposure. We will investigate MLCK activation and tight junction integrity in IL-6 knock out mice and wild type mice given an IL-6 neutralizing antibody. Finally, we will examine how IL-6 affects the MLCK activation signaling by examining the activation of MLCK regulators PKC, Src kinase, and Rho kinase. Finally, activation of activation of myosin light chain phosphatase (MLCP) will be assessed to investigate whether IL- 6 mediates its activation to a greater degree after the combined injury. These studies will help gain an understanding for how even acute ethanol in combination with burn injury can result in decreased barrier function that has been clinically observed and can result in increased morbidity and mortality.

6. 摘要 乙醇是创伤性损伤（包括烧伤）的常见因素。我们实验室之前的研究表明，乙醇会增加烧伤小鼠的肺部和胃肠道炎症以及感染的易感性；然而，这些反应的机制尚不完全清楚。此外，烧伤后，细菌及其产物会从肠腔渗漏到血液中。这些细菌可以扩散到全身，导致肺部损伤、急性肺损伤、败血症和死亡。乙醇与烧伤联合使用已被证明比单独使用任何一种损伤更能降低肠道屏障功能。长 (210 kDa) 平滑肌肌球蛋白轻链激酶 (MLCK) 是一种对上皮紧密连接维持很重要的酶，与烧伤或单独暴露于乙醇后的屏障改变有关。有了这些知识，我们假设急性乙醇暴露和烧伤的结合会导致肠屏障功能障碍的增加，这是由于白细胞介素 6 介导的肌球蛋白轻链激酶的激活所致。为了检验这一假设，提出了三个目标：确定 1) 肌球蛋白轻链激酶激活升高是否介导接受急性乙醇和烧伤治疗的小鼠的肠道通透性改变，2) 白细胞介素 6 (IL-6) 激活肌球蛋白轻链激酶，3) IL-6 在乙醇暴露和烧伤后介导 MLCK 激活。将荧光标记的蛋白质从回肠腔移动到血流，并进行紧密连接蛋白质定位的免疫荧光染色，以研究肠道屏障功能和完整性。 MLCK 激活将通过蛋白质印迹分析来确定。 MLCK 的抑制和敲除也将用于检查该分子在急性乙醇和烧伤暴露后对肠道通透性的作用。我们将研究 IL-6 敲除小鼠和给予 IL-6 中和抗体的野生型小鼠的 MLCK 激活和紧密连接完整性。最后，我们将通过检查 MLCK 调节因子 PKC、Src 激酶和 Rho 激酶的激活来研究 IL-6 如何影响 MLCK 激活信号传导。最后，将评估肌球蛋白轻链磷酸酶(MLCP)的激活，以研究联合损伤后IL-6是否更大程度地介导其激活。这些研究将有助于了解即使是急性乙醇与烧伤相结合也会导致临床观察到的屏障功能下降，并导致发病率和死亡率增加。