晚期氧化蛋白产物通过上调NADPH氧化酶/ROS诱导小肠上皮细胞EMT参与腹腔开放肠空气瘘形成的机制研究

The knowledge, indication, and treatment of open abdomen have undergone dramatic improvements during the past two decades and emerged as a promising approach for managing trauma and non-trauma abdominal emergencies, such as intra-abdominal infection, abdominal compartment syndrome or severe acute pancreatitis. However, open abdomen with concomitant enteroatmospheric fistula(EAF) remains a challenging surgical nightmare, which is associated with high mortality and mobility. Previous studies have demonstrated that intestinal epithelial to mesenchymal transition (EMT) and intestinal oxidative stress play an important role in the pathogenesis of spontaneous intestinal fistulae in inflammatory bowel disease. We found that SNAIL1 and SLUG are elevated in tissue around enteroatmospheric fistula after open abdomen. We have recently shown that open abdomen decreased microvascular blood flow of intestine, leading to intestinal hypoxia, oxidative stress and increased AOPP production. We also found increases in EMT markers and ROS production when IECs were co-cultured with AOPP. Therefore, we hypothesized that oxidative stress-mediated EMT induced EAF formation after open abdomen. To test our hypothesis, we use cell culture, growing Mini-Guts ex vivo and EMT lineage-tracing in vivo to investigate the role of EMT in the development of enteroatmospheric fistula after open abdomen. Then we investigate AOPP regulates EMT-like changes and the formation of EAF through NADPH oxidase/ROS signaling pathways. In conclusion, this study would be able to understood the pathogenesis of enteroatmospheric fistula after open abdomen and solve the key problems for organ protection and EAF prevention after open abdomen, facilitating the wide-scale use of open abdomen for treating trauma, abdominal compartment syndrome, intra-abdominal infection, and other abdominal emergencies.

腹腔开放疗法已成为救治腹部战创伤、复杂腹腔感染和腹腔间隙综合征的重要手段。然而继发的肠空气瘘（EAF）成为阻碍其广泛应用的关键性技术难题。众多研究表明肠上皮间质转变（EMT）和氧化应激是肠瘘自发形成的内在机制。本项目前期研究发现EAF瘘口组织EMT转录因子SNAIL1和SLUG表达增加，提示EMT可能参与EAF形成；腹腔开放导致裸露肠管微循环血流量降低、氧化应激和晚期氧化蛋白产物（AOPP）增加。将AOPP与肠上皮细胞共培养，ROS产生增多，EMT相关蛋白表达增加。基于此，我们提出氧化应激介导的EMT是腹腔开放肠空气瘘形成的内在机制。拟利用细胞培养、Mini-Guts构建和体内细胞谱系示踪技术，研究EMT在腹腔开放肠空气瘘发生发展中的作用机制；探讨AOPP通过激活NADPH氧化酶/ROS调控EMT和EAF形成的机制。本课题将阐明腹腔开放肠空气瘘的形成机制，为肠空气瘘预防和治疗提供新的思路。

腹腔开放疗法已成为救治腹部战创伤、复杂腹腔感染和腹腔间隙综合征的重要手段。然而继发的肠空气瘘成为阻碍其广泛应用的关键性技术难题。吻合口瘘是腹部外科术后常见的严重并发症，也是一直困扰胃肠外科医生的棘手难题。众多研究表明肠道菌群、氧化应激和EMT是肠瘘或吻合口瘘形成的内在机制。基于此，我们提出肠道菌群失调和氧化应激介导的EMT是腹腔开放肠空气瘘或腹部外科术后吻合口瘘形成的内在机制。本项目建立肠道类器官培养方法和应用体系，并初步探索其在肠瘘研究中的价值，为未来进一步研究肠道菌群与肠道相互作用和胃肠恶性肿瘤机制研究奠定了良好的基础。分析了腹腔高压后肠道菌群变化及其在继发肝脏和肠管损伤中的机制研究。研究了腹腔开放后肠管氧化应激变化，并探讨了可注射原位水凝胶对腹腔开放后氧化应激损伤的保护作用。统计分析本中心腹部外科术后吻合口瘘的危险因素，建立术前列线图预测模型和术后早期诊断方法。建立腹部外科术后吻合口瘘动物模型，供后期吻合口瘘的机制研究。综上所述，本项目阐明肠道细菌、氧化应激和EMT在肠空气瘘或吻合口瘘形成中的作用机制，为肠空气瘘和吻合口瘘的预防和治疗提供新的手段。项目资助发表SCI论文1篇，北大核心期刊论文9篇，培养硕士研究生8名，剩余经费将用于细胞谱系示踪研究。