腺泡细胞焦亡及其HIF/Inflammasome/Caspase-1调控通路在急性胰腺炎中的作用及机制研究

The severe local inflammation caused by acute pancreatitis (AP) leads to a large number of acinar cells sterile death. A great deal of previous investigations at home and abroad mainly focused on the relations between necrosis and apoptosis. The central feature of necrotic cell death is the rupture of the cell membrane with the release of pro-inflammatory intracellular contents into extracellular compartment, which are known as damage associated molecular patterns (DAMPs), triggering the immune response. While apoptosis, the first well-recognized programmed cell death, has been evidently demonstrated to contribute to attenuate the inflammation because of the intact cell membrane during cell death. However, another new type of programmed cell death named "pyroptosis", which was used to be confused with apoptosis, has been found and identified. Because of some shared characteristics such as nuclear condensation and DNA fragmentation determined by positive TUNEL assay, pyroptosis was not initially distinguished from apoptosis. However, in contrast to apoptosis, pyroptosis is induced by the activation of inflammasome and maturation of caspase-1 resulting into pore formation in the cell membrane, cell swelling, release of intracellular contents as well as secretion of interleukin-1β (IL-1β) and IL-18. According to the mechanism, characteristics and outcome of the new cell death type, pyroptosis is used to describe the inherently caspase 1-dependent pro-inflammatory programmed cell death and thereby rapidly become the most attractive areas of inflammation. And many significant research achievements have been reported in the prestigious journals like <Nature>, <Science> and <Cell>. In our previous study on pancreatic acinar cell morphology during AP, there were many dying cells showing the combined characteristics of apoptosis (nuclear condensation) and necrosis (rupture of the cell membrane) observed by electron microscope, which were closely consistent with pyroptosis. However, in the fields of AP, there were still no pyroptosis-related investigations at home and abroad. Moreover, the type and degree of acinar cell death are considered to be directly relevant to the prognosis of AP. So this study indicates a valuable scientific prospect in the exploration of pyroptosis and its potential HIF/Inflammasome/Caspase-1 pathway in pancreatic acinar cells during AP.

焦亡是一种新的细胞"程序性死亡"方式，其与凋亡同样出现核固缩、TUNEL染色阳性，在既往研究中两者未被区分；但焦亡却伴有细胞膜破损、炎症因子释放，可加重炎症反应。因此焦亡对炎症疾病的研究尤为重要，一经证实迅速成为国际炎症领域最新的研究热点，近两年《Nature》、《Science》、《Cell》等权威杂志争相报道此类研究。我们前期实验中应用电镜观察急性胰腺炎（acute pancreatitis, AP）腺泡细胞形态，发现许多细胞既有核固缩又有细胞破裂，非常符合焦亡特点，希望查找资料证实，但国内外关于腺泡细胞焦亡的研究至今仍是空白。腺泡细胞最终的死亡方式是焦亡还是凋亡，关系到是促炎还是抑炎，对AP病情至关重要；因此设计本课题期望在验证我们前期实验发现的基础上，研究AP腺泡细胞焦亡机制及HIF/Inflammasome/Caspase-1的调控作用，观察高压氧对细胞焦亡及AP病情的影响。

急性胰腺炎（acute pancreatitis, AP）在我国有较高的发病率，是普外科常见急腹症之一，其中重症急性胰腺炎（severe acute pancreatitis, SAP）又具有近30%的死亡率，因此针对其发病机制的研究具有重要的理论意义和临床价值。胆道结石、酗酒和高血脂等致病因素诱发AP后，胰腺腺泡细胞首先发生损伤，释放的胰酶、细胞因子及细胞崩解产物募集并激活了免疫细胞，使炎症反应级联放大。因此，在AP早期，炎症局部胰腺腺泡细胞的损伤程度及死亡方式决定了后续炎症反应的轻重。之前普遍认为，胰腺腺泡细胞如果以凋亡方式死亡，则减少胰酶和炎症因子的释放，但随着人们对细胞死亡方式认识的逐渐深入，细胞的死亡方式往往是细胞死亡过程的一个阶段，很难存在凋亡与坏死之间的绝对分界。更重要的是，近期发现了一种新的细胞“程序性死亡”方式——焦亡，焦亡细胞的细胞膜破损，伴有内容物和细胞因子释放，促进炎症反应，但焦亡与凋亡同样出现核固缩、TUNEL染色阳性，既往研究中两者未被区分。在本研究中，我们首先证实了AP发病机制中存在腺泡细胞焦亡，并且焦亡细胞越多AP病情越重。我们证实AP病程中腺泡细胞焦亡最显著的时间点是24h。在参与AP腺泡细胞焦亡的诸多炎症小体中，NLRP3功能最强。钙离子超载和ATP耗竭促使NLRP3和ASC表达上调，caspase-1活化增加, IL-1和IL-18含量增加，加重了急性胰腺炎病情。抑制腺泡细胞焦亡有助于改善AP病情。