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

描述（由申请人提供）：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对感染的易感性;然而，这些反应的机制尚不完全清楚。此外，在烧伤后，细菌及其产物从肠腔泄漏并进入血流。这些细菌可以分散到全身，导致肺损伤、急性肺损伤、败血症和死亡。乙醇与烧伤联合使用比单独使用更能降低肠屏障功能。长（210 kDa）平滑肌肌球蛋白轻链激酶（MLCK），一种重要的上皮紧密连接的维护酶，已牵连在烧伤或乙醇暴露后的屏障改变。有了这些知识，我们假设急性乙醇暴露和烧伤的组合导致肠屏障功能障碍的增加，由于白细胞介素-6介导的肌球蛋白轻链激酶的激活。为了验证这一假设，提出了三个目标：确定1）在接受急性乙醇和烧伤治疗的小鼠中，肌球蛋白轻链激酶激活是否升高介导肠通透性改变，2）白细胞介素-6（IL-6）激活肌球蛋白轻链激酶，3）IL-6介导乙醇暴露和烧伤后MLCK激活。将进行荧光标记的蛋白质从回肠腔中移出至血流沿着，并进行免疫荧光染色以进行紧密连接蛋白定位，以研究肠屏障功能和完整性。MLCK活化将通过蛋白质印迹分析测定。MLCK的抑制和敲除也将被用于检查该分子在急性乙醇和烧伤损伤暴露后对肠通透性的作用。我们将研究给予IL-6中和抗体的IL-6敲除小鼠和野生型小鼠中的MLCK激活和紧密连接完整性。最后，我们将通过检测MLCK调节剂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）的活化以研究IL- 6是否在复合损伤后更大程度地介导其活化。这些研究将有助于了解即使急性乙醇与烧伤联合使用也会导致屏障功能降低，这在临床上已经观察到，并可能导致发病率和死亡率增加。 公共卫生相关性：在美国，伤害仍然是生命最初四十年的主要死亡原因，超过了包括心脏病和癌症在内的所有其他死亡原因，每年造成数百万起致残伤害，每年造成的损失超过1300亿美元。乙醇是关于损伤的重要复杂因素，如从高达50%的烧伤患者在入院时在其循环中具有可检测水平的乙醇的发现中所见。这些患者中的大多数不是慢性酗酒者，而是急性或酗酒者，与具有类似损伤的非饮酒受试者相比，其发病率和死亡率增加。

DESCRIPTION (provided by applicant): 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. PUBLIC HEALTH RELEVANCE: In the United States, injury remains the primary cause of death during the first four decades of life, outnumbering all other causes of death, including heart disease and cancer, and accounts for millions of disabling injuries per year and an annual cost of over $130 billion. Ethanol is an important complicating factor regarding injury as seen from the finding that up to 50% of burn patients have detectable levels of ethanol in their circulation at the time of admission to the hospital. These patients, the majority of whom are not chronic alcoholics, but rather acute or binge drinkers, suffer from increased morbidity and mortality compared with that of non-alcohol-consuming subjects with similar injuries.