自噬，凋亡，还是坏死？TLR4通路介导急性胰腺炎细胞死亡调控机制研究

The mortality of severe acute pancreatitis (SAP) remains high and challenging doctors worldwide. The severity of acute pancreatitis is paralleled with the predominance of a given type of cell death, and inflammatory attack in AP could be alleviated by regulating the different types of cell death development. Autophagy, apoptosis and necrosis, all of which are typical cell death involved in AP, but the potential regulatory mechanism between the three and the relation and effector for the prognosis of AP remain unclear. Based on our preliminary results, which evidenced pancreatic apoptosis was significantly different between mice with or without the expression of Toll like receptor 4 (TLR4), and a big amount of autophagy-like vesicles could be detected in cytoplasm at specific time points of AP, we proposed that TLR4 might potentially be involved in the regulation of three types of cell death in AP. This project focuses on the potential role of TLR4 in regulating autophagy, apoptosis and necrosis in the development of AP, with comprehensively apply multiple mice strains and multiple stable cell lines with key regulatory gene of inflammatory or cell death pathways knockout/knockdown, to intensively study and analyze the role of TLR4 signaling in possible predisposing or regulating different types of cell deaths in the development of AP. This study will provide new insights for understanding the pathogenesis of AP, and provide research evidences for further translational diagnosis and treatment of AP.

重症急性胰腺炎(SAP)病死率仍居高不下,是世界范围内的医学难题。急性胰腺炎(AP)的严重程度与细胞死亡方式相关,调控胰腺细胞死亡方式将影响AP的发展与转归。自噬、凋亡与坏死作为特征性的细胞死亡方式均参与了AP病程,但决定AP细胞死亡方式取向的调控机制,以及影响AP转归的机理不明。申请者基于前期发现：TLR4作为炎症反应核心蛋白,其缺失与野生小鼠株在诱导AP后,胰腺细胞凋亡坏死存在显著差异；在特定时点，胰腺腺泡细胞超微结构中可见大量类似自噬空泡化的结构改变,提出TLR4可能参与AP病程中多种细胞死亡方式的取向调控。本课题以TLR4信号介导自噬,凋亡,坏死为切入点,联合采用多株炎症-细胞死亡通路关键分子基因缺失小鼠及基因稳定沉默细胞系,深入探讨AP中TLR4信号通路与胰腺细胞死亡的调控规律及对AP病程影响，结果将对AP发病机理提出新的认识，并为AP/SAP的预防及治疗提供转化思路和实验依据。

【背景及目的】重症急性胰腺炎（SAP）起病急骤，病死率高达50%；SAP由急性胰腺炎（AP）发展而来，AP的基本病理改变是腺泡细胞死亡，导致胰腺局部及全身炎症反应。凋亡和坏死作为腺泡细胞两种主要的死亡方式，两者间的平衡与AP预后密切相关,但AP中调控凋亡和坏死的机制未完全阐明。新进发现自噬作为一种新的细胞死亡方式，参与了AP的发生发展，但自噬与AP腺泡细胞死亡之间的关系尚不明确；TLR4（Toll-like receptor 4）作为天然免疫系统中炎症反应的起始点，在AP初期介导炎性细胞因子产生，但在胰腺组织中TLR4是否可以调控自噬，进而影响AP的进展，以及TLR4调控胰腺组织自噬的信号通路，尚无研究。本研究以 TLR4 介导细胞死亡（自噬,凋亡,坏死）为切入点,深入探讨AP中TLR4通路与胰腺细胞死亡的调控规律及对 AP 病程影响，拟为临床治疗提供新的思路及实验依据。.【研究内容】①观察自噬在雨蛙素（cerulein，Cer）和雨蛙素联合脂多糖（Cer+LPS）诱导小鼠AP中的表达差异，探讨自噬与AP严重程度的关系；②探讨自噬抑制剂3-甲基腺嘌呤（3-MA）对Cer+LPS诱导的AP自噬及腺泡细胞凋亡的影响，进一步明确自噬在AP发生发展中的作用，探索抑制自噬对AP的治疗作用；③探讨Toll样受体4（toll-like receptor 4，TLR4）对AP自噬的调控作用及其机制。.【结果】.SAP中自噬水平明显升高且自噬流受阻更明显。3-MA显著降低了AP的严重程度，并抑制了自噬的诱导水平，降低了自噬流；3-MA明显上调胰腺组织的凋亡相关基因水平，并导致胰腺组织细胞凋亡增加；TLR4缺失显著降低AP的严重程度，并下调胰腺细胞的自噬诱导水平。TLR4调控自噬可能依赖于AMPK-mTOR-ULK1信号通路。.【结论】.自噬参与了AP的发生发展，AP中自噬诱导水平明显上调，但自噬性降解受阻，导致自噬流受损； SAP中自噬流受损的程度较MAP更重；3-MA可通过抑制自噬的诱导水平，缓解自噬流受阻，显著减轻SAP的严重程度。TLR4缺失对SAP发挥了保护性作用，通过下调自噬诱导水平可能是其机制之一，且TLR4调控自噬水平依赖于AMPK-mTOR-ULK1信号通路。3-ma联合机体炎症免疫调节可为AP的早期治疗提供思路。