晚期氧化蛋白产物通过MLCK-MLC信号通路诱导肠粘膜屏障损伤的作用机制研究

The integrity of intestinal mucosal barrier is essential for health, and intestinal mucosal barrier injury has been identified to be involved in various intestinal diseases and clinical critical diseases. Therefore, to explore the mechanism of intestinal barrier injury and the potential therapeutics is vital for these diseases. Our previous study found that advanced oxidation protein products (AOPPs) accumulated in intestinal lesion tissues of patients with Crohn’s disease, and induced epithelial-mesenchymal transition of intestinal epithelial cell, which suggested that AOPPs played a role in intestinal barrier injury via the transcellular pathway. However, whether AOPPs have a regulatory effect in disrupting the intestinal barrier integrity via the para cellular pathway remains unknown. Hence, to further determine the role and underlying mechanism of AOPPs in intestinal barrier injury, following work will be performed: through cell and animal experiments, to identify the role of AOPPs in regulating the expression and distribution of intestinal epithelial cell tight junction related proteins, the cell morphological changes and cytoskeleton rearrangement; to verify the effect of AOPPs on the permeability of intestinal mucosal barrier; to explore the molecular mechanism underlying the disruption of intestinal epithelial barrier integrity triggered by AOPPs, and determine whether the NADPH dependent ROS-mediated MLCK signaling contribute to it. Our study will further supplement the pathological mechanism of intestinal barrier injury, and thus provide a new way to prevention and treatment of intestinal diseases and clinical critical diseases.

肠粘膜屏障的完整性是机体保持健康的重要基础，多种肠道疾病及临床危重症与肠粘膜屏障损伤有关，因此探索肠粘膜屏障损伤的机制、寻求维护肠粘膜屏障功能的有效药物对这些疾病至关重要。申请人前期工作发现，晚期氧化蛋白产物（AOPPs）在克罗恩病患者肠道病变组织中显著沉积，AOPPs可诱导肠上皮细胞间质转化，提示AOPPs可通过跨细胞途径影响肠上皮屏障功能。但是AOPPs对细胞旁途径介导的肠上皮屏障的调控作用尚不清楚，据此，本课题拟开展如下工作：通过体内外实验研究AOPPs干预后肠上皮细胞紧密连接蛋白表达及分布的改变、细胞形态如微绒毛及细胞骨架的改变；检测AOPPs干预后肠上皮屏障通透性的变化；探讨AOPPs通过NADPH-ROS介导的肌球蛋白轻链激酶（MLCK）信号通路在肠上皮屏障功能调控中的作用。项目完成将补充和完善肠粘膜屏障损伤的机制，为临床寻求肠道疾病及临危重病症的防治奠定理论基础、提供新途径。

肠粘膜屏障是克罗恩病（CD）等多种消化系统疾病的重要靶标，肠上皮屏障损伤是导致肠道甚至全身多种炎症性疾病和或免疫性疾病的重要环节。肠上皮紧密连接（tight junction，TJ）是肠粘膜屏障的结构基础，其结构缺陷可导致肠粘膜屏障的损伤。因此，探寻肠上皮TJ破坏的发病机制、寻找更有效的治疗靶点是CD研究领域的热点。在本项目中，我们证实了晚期氧化蛋白产物（AOPPs），一种氧化应激的标志物和新的致炎因子，可以通过减少TJ蛋白的表达和破坏TJ结构的完整，诱导肠上皮屏障的损伤。首先，本项目通过临床样本实验发现AOPPs在CD患者肠道病变组织显著沉积，而TJ蛋白在CD中表达明显减少；并且AOPPs在CD中的沉积与TJ蛋白的表达呈负相关。本项目进一步通过构建单层上皮屏障模型和动物实验，观察AOPPs对TJ、上皮屏障结构和功能及其调控信号通路的作用。我们发现AOPPs可以通过减少肠上皮细胞间TJ蛋白的表达，破坏肠上皮TJ结构的完整性，增加肠上皮屏障的通透性，诱导肠粘膜屏障结构和功能的损伤。同时，本项目阐明了AOPPs通过其受体途径，激活NOX2氧化酶依赖的活性氧生成，活化ERK-MLCK/MLC信号轴，从而参与破坏肠上皮屏障。我们的结果表明以AOPPs这一致病因子为关键的分子级联网络可能是维护肠上皮屏障完整性的治疗靶点，也为防治克罗恩病等慢性疾病提供了科学理论依据和新的干预途径。