Interaction of endocytic vacuoles with cellular organelles as a trigger for the cell damage in acute pancreatitis.

急性胰腺炎是一种常见的危及生命的炎症性疾病。过量的酒精摄入和胆结石是这种衰弱状况的重要危险因素。受这种疾病影响的人口比例正在迅速增加。在急性胰腺炎的情况下，通常有助于消化肠道中食物的消化酶在胰腺内变得活跃，并且该器官本身“消化”。胰蛋白酶是参与这一过程的关键消化酶。在正常健康的胰腺细胞中，胰蛋白酶的前体（胰蛋白酶原）被包装在小颗粒（分泌颗粒）中。这种前体不能分解蛋白质或对细胞造成损害。当消化过程受到刺激时，由于含有胰蛋白酶原的分泌颗粒与细胞表面膜的分泌融合，胰蛋白酶原从细胞中释放出来。正常情况下，胰蛋白酶原只有在到达肠时才被激活，在肠中胰蛋白酶完成其分解（消化）膳食蛋白质的功能。然而，当我们重建导致急性胰腺炎的条件时，胰蛋白酶原在胰腺细胞内被不适当地激活。我们认为这是因为在分泌过程中并非所有的胰蛋白酶原都从细胞中释放出来，其中一些被带回细胞中，形成称为内吞空泡的囊泡（以某种方式获得激活胰蛋白酶原并形成活性的潜在破坏性胰蛋白酶的能力）。我们最近发现，内吞空泡可以破裂并将其内容物释放到细胞内部。我们还发现，空泡可以与细胞表面融合，并将其内容物输送到细胞外空间。这些重要的未发表的观测结果构成了本申请的基础。我们假设空泡的破裂和/或空泡与细胞表面的融合对于引发急性胰腺炎的细胞损伤是必不可少的（因为这些过程应该允许胰蛋白酶破坏重要的细胞内和细胞外蛋白质）。我们想验证一下这个假设。另一个重要而未解决的问题是“胰蛋白酶原如何在内吞空泡内被激活？“我们认为这是因为含有胰蛋白酶原的空泡与负责细胞内消化的细胞隔室（细胞器）相互作用。需要细胞内消化来回收细胞部分和蛋白质。在负责细胞内消化的隔室中发现可以破坏蛋白质的分子，并且它们原则上可以激活胰蛋白酶原。因此，我们计划研究的相互作用的囊泡，回收胰蛋白酶原进入细胞的细胞器负责细胞消化（内体，溶酶体和自噬体）。我们将试图确定这些细胞器中的哪一个有助于激活胰蛋白酶原，以及细胞器相互作用的过程是如何启动的。胰蛋白酶原激活不是细胞损伤的唯一标志。其他指标包括对负责能量产生的细胞区室（线粒体）的损害和对维持细胞形状的结构（细胞骨架）的损害。我们将研究胰蛋白酶原激活与其他细胞损伤指标，以确定导致急性胰腺炎细胞死亡的事件顺序。最后，我们将研究胰蛋白酶在一个细胞中产生的损伤如何传播并影响器官中的其他细胞。了解导致消化酶激活的过程将有助于急性胰腺炎治疗的发展。为了加速治疗的发展，在项目过程中产生的所有信息，分子探针和方法创新将直接进入利物浦NIHR胰腺生物医学研究单位进行的药物发现转化研究计划。

Acute pancreatitis is a frequent and life-threatening inflammatory disease. Excessive alcohol intake and gallstones are important risk factors for this debilitating condition. The proportion of the population affected by this disease is increasing rapidly. In conditions of acute pancreatitis, digestive enzymes, which normally help to digest food in the intestine, become active inside the pancreas and this organ "digests" itself. Trypsin is a key digestive enzyme involved in this process. In normal healthy cells of the pancreas, a precursor of trypsin (trypsinogen) is packaged inside small granules (secretory granules). This precursor cannot breakdown proteins or cause damage to the cells. When the process of digestion is stimulated, trypsinogen is released from the cells as a result of secretion - fusion of the secretory granules containing trypsinogen with the surface membrane of the cell. Normally trypsinogen becomes activated only when it reaches intestine where trypsin fulfils its function of breaking down (digesting) dietary proteins. However when we recreated conditions that lead to acute pancreatitis, trypsinogen was activated inappropriately inside the cells of the pancreas. We think that this happens because not all trypsinogen is released from the cell during secretion and some of it is taken back into the cell in vesicles termed endocytic vacuoles (which somehow acquire the ability to activate trypsinogen and to form the active potentially damaging trypsin). We recently discovered that the endocytic vacuoles can rupture and release their contents into the interior of the cell. We also found that the vacuoles can fuse with the cell surface and deliver their contents into the extracellular space. These important unpublished observations form the basis of this application. We hypothesise that the rupture of the vacuoles and/or the fusion of vacuoles with the cell surface are essential for the cell damage that initiates acute pancreatitis (because these processes should allow trypsin to destroy vital intracellular and extracellular proteins). We would like to test this hypothesis. Another important and unresolved question is "How the trypsinogen is activated inside the endocytic vacuoles?" We propose that this happens because the trypsinogen-containing vacuoles interact with cell compartments (organelles) that are responsible for intracellular digestion. Intracellular digestion is needed to recycle the cell parts and proteins. Molecules that can break proteins are found in the compartments responsible for intracellular digestion and they can in principle activate trypsinogen. We are therefore planning to study the interaction of vesicles that retrieve trypsinogen into the cell with organelles responsible for cellular digestion (endosomes, lysosomes and autophagosomes). We will try to determine which of these organelles helps to activate trypsinogen and how the process of organelle interaction is initiated. Trypsinogen activation is not the only hallmark of cell injury. Other indicators include damage to the cellular compartment responsible for energy production (mitochondria) and damage to structures maintaining cell shape (cytoskeleton). We will investigate trypsinogen activation together with the other indicators of cell damage to determine the sequence of events leading to cell death in acute pancreatitis. Finally we will study how damage produced by trypsin in one cell can propagate and affect other cells in the organ. The understanding of the processes leading to activation of digestive enzymes will facilitate the development of the treatment against acute pancreatitis. To accelerate the development of the treatment all information, molecular probes and methodological innovations generated in the course of the project will feed directly into the translational research programme of drug discovery undertaken by the Liverpool NIHR Pancreas Biomedical Research Unit.