Molecular mechanisms mediating colonic anastomotic leakage.

本研究旨在确定前列腺素信号传导在吻合口漏中的作用，并测试支持愈合途径的干预策略。在腹部手术期间，外科医生对内脏器官的术中处理不可避免地施加手术创伤，并且所产生的组织损伤可以影响正常的器官功能。我们的小组在研究几种外科创伤引起的疾病的病理机制方面有着丰富的经验，包括术后肠梗阻、腹腔内粘连形成和吻合口瘘。在本计画中，我们将探讨严重干扰结肠手术病患恢复的吻合口漏的分子机制。结直肠吻合口瘘（CAL）是一种可怕的手术并发症，占结直肠手术后死亡人数的三分之一。CAL已被认为是一个复杂的过程，涉及不同的细胞反应。前列腺素（PG）是由花生四烯酸通过环氧合酶（考克斯）产生的，并且对于调节伤口愈合过程至关重要。一致地，已知在临床环境中抑制考克斯活性可显著增加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小鼠CAL，并鉴定PGE2受体EP2和EP4为CAL的潜在介导剂。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由各种细胞类型表达并影响许多细胞过程，需要进行详细的研究来确定CAL的分子机制，以允许对其病理进行询问。在这项研究中，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）在两种不同的CAL模型中研究内源性PGE2降解途径、EP 2和EP 4受体激动剂的抑制以及细胞特异性EP 2和EP 4信号传导的抑制，以鉴定CAL预防中潜在的新途径。iii）通过基于UPLC-MS的氧化脂质组测定对人类吻合愈合者和泄漏者血液样品进行转化分析，以确定COX衍生的介质模式和与经历结肠直肠吻合手术的人类中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提供新的见解愈合过程中的小鼠吻合和CAL的信号通路，重点是PGE2信号。本文生成的数据包括在单细胞水平上的全面转录表征，脂质组学分析，空间转录组学和CAL中的干预策略。总之，这些数据将有助于开发靶向药物，以防止未来的CAL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.