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

描述（由申请人提供）：乙醇是创伤性损伤的常见因素，包括烧伤。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，细菌及其产物会从肠腔中泄漏到血液中。这些细菌可以扩散到全身，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任何一种损伤都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，与烧伤或乙醇暴露后的屏障改变有关。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道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。为了验证这一假设，我们提出了三个目的：确定1)肌球蛋白轻链激酶激活升高是否介导急性乙醇和烧伤治疗小鼠肠通透性的改变，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的激活。将荧光标记蛋白从回肠腔转移到血液中，同时进行紧密连接蛋白定位的免疫荧光染色，以研究肠屏障功能和完整性。MLCK激活将通过Western blot分析确定。我们还将利用MLCK的抑制和敲除来研究该分子在急性乙醇和烧伤暴露后对肠道通透性的作用。我们将在IL-6敲除小鼠和给予IL-6中和抗体的野生型小鼠中研究MLCK的激活和紧密连接的完整性。最后，我们将通过检测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。最后，我们将评估肌球蛋白轻链磷酸酶（MLCP）的激活情况，以研究IL- 6是否在联合损伤后更大程度地介导其激活。这些研究将有助于了解急性乙醇合并烧伤如何导致临床观察到的屏障功能下降，并可能导致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.