Molecular mechanisms mediating colonic anastomotic leakage.

本研究旨在确定前列腺素信号在吻合口瘘中的作用，并测试支持愈合途径的介入策略。在腹部手术中，外科医生术中对内脏器官的处理不可避免地会产生外科创伤，由此造成的组织损伤会影响正常的器官功能。本组对术后肠梗阻、腹内粘连、吻合口瘘等外科创伤性疾病的病理机制有丰富的研究经验。在本项目中，我们将探讨严重影响结肠手术患者康复的吻合口瘘的分子机制。结直肠吻合口瘘（CAL）是一种可怕的手术并发症，占结直肠手术死亡总数的三分之一。CAL被认为是一个复杂的过程，涉及多种细胞反应。前列腺素（PG）由花生四烯酸通过环氧化酶（COX）产生，对调节伤口愈合过程至关重要。一直以来，在临床环境中抑制COX活性被认为会大大增加CAL，因此在临床检查中经常被规避。为了评估驱动CAL的分子途径，我们通过转录谱分析了小鼠CAL，并确定了PGE2受体EP2和EP4是CAL的潜在介质。我们假设EP2或EP4信号调节吻合口愈合，这些途径的干扰可能介导CAL。由于EP2和EP4在各种细胞类型中表达，并影响许多细胞过程。有必要进行详细的研究，以确定CAL的分子机制，以便对其病理进行讯问。在本研究中，我们有三个主要目标：i)通过单细胞RNAseq，解吸电喷雾质谱成像（DESI）和空间转录组学鉴定CAL中PGE2和细胞因子产生的细胞来源和位置。ii)研究两种不同CAL模型中PGE2内在降解途径、EP2和EP4受体激动剂的抑制作用以及细胞特异性EP2和EP4信号的抑制作用，以确定预防CAL的潜在新途径。iii)通过基于uplc - ms的氧脂质面板分析法对人类吻合口愈合者和泄密者血液样本进行翻译分析，以确定cox衍生介质模式及其与结肠直肠吻合口手术患者不同愈合结果的潜在关联。总的来说，该项目产生的数据集将为愈合和CAL期间小鼠吻合的信号通路提供新的见解，重点是PGE2信号。本文生成的数据包括单细胞水平的综合转录特征、脂质组学分析、空间转录组学和CAL的干预策略。这些数据将有助于开发靶向药物来预防CAL的预防。

This study aims to identify the role of prostaglandin signalling in anastomotic leakage and to test interventional strategies to support healing pathways. During abdominal surgery, the surgeon's intraoperative handling of visceral organs unavoidably exerts a surgical trauma, and the resulting tissue damage can affect regular organ function. Our group has extensive experience studying the pathological mechanisms of several surgical trauma-induced disorders, including postoperative ileus, intra-abdominal adhesion formation and anastomotic leakage. In this project, we will explore the molecular mechanisms of anastomotic leakage that significantly disturb colonic surgery patients' recovery. Colorectal anastomotic leakage (CAL) is a dreaded surgical complication and corresponds to one-third of all deaths after colorectal surgery. CAL has been recognised as a complex process involving diverse cellular responses. Prostaglandins (PG) are produced from arachidonic acid via cyclooxygenases (COX) and are crucial for regulating wound healing processes. Consistently, inhibition of COX activity in a clinical setting is known to augment CAL considerably and is therefore often circumvented in the clinical workup. To assess the molecular pathways driving CAL, we analysed CAL in mice through transcriptional profiling and identified PGE2 receptors EP2 and EP4 as potential mediators of CAL. We hypothezised that EP2 or EP4 signalling regulates anastomotic healing and disturbances of these pathways are likely to mediate CAL. Since EP2 and EP4 are expressed by various cell types and influence numerous cellular processes, a detailed investigation is necessary to identify the molecular mechanisms of CAL to allow interrogation of its pathology. In this study, we have three main objectives: i) Identification of the cellular source and location of PGE2 and cytokine production in CAL by single-cell RNAseq, desorption electrospray mass spectrometry imaging (DESI) and spatial transcriptomics. ii) To study the inhibition of intrinsic PGE2 degrading pathways, EP2 and EP4 receptor agonists and inhibition of cell-specific EP2 and EP4 signalling in two different CAL models to identify potentially novel pathways in CAL prevention. iii) Translational analyses of human anastomotic healers and leakers blood samples by UPLC-MS-based oxylipin panel assay to determine COX-derived mediator patterns and potential association with different healing outcomes in humans undergoing colorectal anastomotic surgery. Overall, the datasets yielded by this project will provide novel insight into the signalling pathways of murine anastomosis during healing and CAL with a focus on PGE2 signalling. The data generated herein include comprehensive transcriptional characterisation at the single cell level, lipidomic profiling, spatial transcriptomics and interventional strategies in CAL. Together, these data will help to develop targeted drugs to prevent CAL prevention in the future.