GUT BARRIER DYSFUNCTION: THE TRIGGER FOR CACHEXIA IN APCMIN/+ MICE

这个子项目是许多研究子项目中利用 资源由NIH/NCRR资助的中心拨款提供。子项目和 调查员(PI)可能从NIH的另一个来源获得了主要资金， 并因此可以在其他清晰的条目中表示。列出的机构是 该中心不一定是调查人员的机构。 癌症恶病质是一种全身衰弱的状态，在胃肠道和肺癌中更为常见；恶病质占结肠癌死亡的30%-50%。因此，预防恶病质是改善结肠癌患者临床预后的重要目标。对肌肉萎缩的细胞机制的研究表明，恶病质是由炎症信号启动的，然后激活ATP依赖的泛素依赖的蛋白酶体降解蛋白质，导致肌肉质量的损失。 了解整合炎症信号和肌肉蛋白分解的调节机制对于理解恶病质是至关重要的。PI目前拥有由国家癌症研究所资助的RO1赠款，用于研究炎性细胞因子IL-6对恶病质ApcMin/+小鼠肌肉蛋白质合成和降解的调节以及肌肉线粒体功能。ApcMin/+小鼠慢性低水平炎症的来源尚未完全确定，但被认为与对肠道和结肠肿瘤的免疫反应有关，也可能涉及肿瘤细胞直接分泌细胞因子。我们目前的资金没有解决的一个问题是全身炎症的原因，这种炎症被认为是启动ApcMin/+小鼠骨骼肌萎缩的原因。 肠屏障功能障碍(GBD)是指肠上皮细胞之间的紧密连接不再形成阻止细菌渗入肠粘膜下层和肌层的不渗透屏障。8这可导致内毒素血症(血液中的细菌细胞壁碎片)或败血症(血液中的活菌)，导致多器官功能障碍综合征(MODS)。IL-6基因敲除的大鼠不会发生GBD。27我们最近的研究表明，IL-6基因敲除也可以抑制恶病质的发展，并且IL-6的过度表达会在ApcMin/+小鼠中诱导恶病质。5我们的初步研究表明，30周龄的ApcMin/+小鼠的一部分血液循环内毒素检测呈阳性，肠系膜淋巴结处的细菌检测呈阳性。目前尚不清楚GBD合并低水平内毒素血症或败血症，加上IL-6表达增加，是否是促进ApcMin/+小鼠肿瘤形成、肿瘤生长和/或恶病质的重要炎症来源。 目前这项建议的总体目的是确定肠道屏障功能障碍是否是ApcMin/+小鼠恶病质的主要原因。就我们所知，工作假说完全是新奇的。我们假设恶病质是由ApcMin/+小鼠体内内毒素(细菌脂多糖或完整细菌)通过胃肠道屏障渗漏引起的炎症增加引起的。此外，我们假设，在ApcMin/+和ApcMin/+x Il-6-/-小鼠中，循环中IL-6的升高将是诱导内毒素通过胃肠道屏障渗漏所必需的。这项建议将约翰·贝恩斯博士在分析生物化学、临床生物化学和新陈代谢方面的经验与卡森博士使用包括ApcMin/+小鼠在内的整个动物模型对生理学的理解结合在一起。这项提议也是一个新的项目，可以使贝恩斯博士的研究专长更接近位于 南卡罗来纳大学。这项工作的完成将有助于更好地了解GBD和IL-6信号在恶病质调节中的作用，并找到治疗和预防恶病质疾病的潜在治疗靶点。该提案将为多年期RO1应用的进一步研究提供必要的初步数据。 目的1.确定ApcMin/+小鼠的肠道屏障功能障碍是否随着肿瘤负荷和恶病质的增加而增加。 目的2.确定炎性细胞因子IL-6是否导致ApcMin/+和ApcMin/+x IL-6-/-小鼠肠屏障功能障碍，并伴随恶病质的发展。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Cancer cachexia is a condition of whole body wasting that is more common with gastrointestinal tract and lung cancers; cachexia accounts for 30-50% deaths with colon cancer. Thus, the prevention of cachexia is an important objective for the improvement of clinical outcomes in colon cancer patients. Studies on cellular mechanisms involved in muscle wasting indicate that cachexia is initiated by inflammatory signaling, which then activates ATP-dependent ubiquitin-dependent proteasomal degradation of protein, causing loss of muscle mass. Understanding the regulatory mechanisms that integrate inflammatory signaling and muscle proteolysis is critical for understanding cachexia. The PI currently has a RO1 grant funded through the National Cancer Institute to examine inflammatory cytokine IL-6 regulation of muscle protein synthesis and degradation, and muscle mitochondrial function in cachectic ApcMin/+ mice. The source of chronic low level inflammation in ApcMin/+ mice is not well established, but is thought to be related to the immune response to intestinal and colon tumors, and may also involve direct tumor cell secretion of cytokines. A question not addressed by our current funding is the cause of the systemic inflammation that is thought to initiate the skeletal muscle wasting in ApcMin/+ mice. Gut barrier dysfunction (GBD) is a condition where tight junctions between intestinal epithelial cells no longer form an impermeable barrier against bacterial infiltration into the submucosa and muscularis mucosa of the intestine.8 This can lead to endotoxemia (bacterial cell wall fragments in blood) or sepsis (viable bacteria in blood), leading to multiple organ dysfunction syndrome (MODS).7, 21 GBD has been well documented in hemorrhage shock models, and is associated with, and dependent on, increased circulating IL-6 levels; rats with knock-out of IL-6 do not develop GBD.27 We have recently shown that knockout of IL-6 also inhibits development of cachexia and that IL-6 over-expression induces cachexia in ApcMin/+ mice.5 Our preliminary studies demonstrate that a subset of 30 week old ApcMin/+ mice test positive for circulating endotoxin in the blood, and bacteria in the mesenteric lymph nodes. It is not known if GBD with low-level endotoxemia or sepsis, combined with increased expression of IL-6, is a significant source of inflammation that promotes tumorigenesis, tumor growth, and/or cachexia in ApcMin/+ mice. The overall purpose of the current proposal is to determine if gut barrier dysfunction is the primary cause of cachexia in ApcMin/+ mice. To the best of our knowledge the working hypothesis is completely novel. We hypothesize that cachexia is initiated by increased inflammation that results from endotoxin (bacterial lipopolysaccharide or intact bacteria) leakage across the gastrointestinal barrier in ApcMin/+ mice. Additionally, we hypothesize that elevated circulating IL-6 will be necessary to induce endotoxin leakage across the gastrointestinal barrier in ApcMin/+ and ApcMin/+ x Il-6-/- mice. This proposal merges Dr. John Baynes experience in analytical biochemistry, clinical biochemistry, and metabolism with Dr. Carson's understanding of physiology using whole animal models, including the ApcMin/+ mouse. This proposal also servers as a novel project that can bring Dr. Baynes's research expertise closer to the colorectal cancer research group at the University of South Carolina. Completion of this work will lead to a better understanding of the role of GBD and IL-6 signaling in regulation on cachexia, and identify potential novel therapeutic targets to both treat and prevent the cachectic condition. This proposal will provide essential preliminary data for further study in a multi-year RO1 application. Aim 1. Determine if gut barrier dysfunction increases with tumor burden and cachexia in the ApcMin/+ mouse. Aim 2. Determine if inflammatory cytokine IL-6 induces gut barrier dysfunction, in concert with the development of cachexia, in ApcMin/+ and ApcMin/+ x IL-6 -/- mice.