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

6.摘要 酒精是包括烧伤在内的创伤性损伤的常见因素。我们实验室以前的研究表明，乙醇增加了烧伤小鼠的肺部和胃肠道炎症以及感染的易感性；然而，这些反应的机制并不完全清楚。此外，烧伤后，细菌及其产物会从肠腔泄漏到血液中。这些细菌可以散布在全身，导致肺损伤、急性肺损伤、败血症和死亡。研究表明，酒精与烧伤合并使用时，肠道屏障功能的降低程度要比单独使用酒精时更大。长(210 KDa)的平滑肌肌球蛋白轻链激酶(MLCK)是维持上皮紧密连接的重要酶，已被认为与烧伤或单纯酒精暴露后屏障的改变有关。有了这些知识，我们假设急性酒精暴露和烧伤的结合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。为了验证这一假说，提出了三个目标：1)肌球蛋白轻链激酶活性升高是否介导了急性酒精和烧伤小鼠肠道通透性的改变；2)白介素6(IL-6)激活了肌球蛋白轻链激酶；3)IL-6介导了酒精暴露和烧伤后MLCK的激活。荧光标记的蛋白质从回肠腔移动到血流以及紧密连接蛋白质定位的免疫荧光染色将被用来研究肠屏障的功能和完整性。MLCK的活性将通过Western印迹分析来确定。MLCK的抑制和敲除也将被用来研究该分子在急性酒精和烧伤暴露后对肠道通透性的作用。我们将研究IL-6基因敲除小鼠和给予IL-6中和抗体的野生型小鼠的MLCK活性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和Rho的激活来研究IL-6对MLCK激活信号的影响。最后，将评估肌球蛋白轻链磷酸酶(MLCP)的激活情况，以探讨IL-6是否在更大程度上介导了其在复合损伤后的激活。这些研究将有助于理解，即使是急性酒精合并烧伤也会导致临床观察到的屏障功能降低，并可能导致发病率和死亡率的增加。

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.