基于"以通为用"理论重症急性胰腺炎内质网应激介导的中性粒细胞凋亡异常的研究

Systemic inflammatory response syndrome (SIRS) is early stage of multiple organ dysfunction syndrome (MODS) caused by severe acute pancreatitis (SAP), which is a critical and serious illness. Therefore, blocking SIRS progress to the MODS is of important clinical significance. Polymorphonuclear neutrophils (PMN), an important indicator of inflammatory response, whose apoptosis is delayed contributes to aggravation of SIRS. In this study, Tongli Gongxia method, as a starting point, and abnormal apoptosis of PMN, as a main line, will be included in studying the molecular mechanisms of abnormal neutrophil apoptosis from endoplasmic reticulum stress-mediated pathway in vitro and in vivo. A variety of cellular and molecular biotechnology will be applied, and the shape of the endoplasmic reticulum of neutrophils, calcium homeostasis, unfolded protein response and other aspects of abnormal endoplasmic reticulum stress-mediated signaling pathway of PMN apoptosis will be observed in this study. The mechanisms of Dachengqi decoction, which is a classical prescription of Tongli Gongxia method, and Emodin in above-mentioned directions will be studied as well. To deeper analyze the molecular mechanisms of delayed apoptosis of neutrophils and abnormal endoplasmic reticulum stress-mediated signaling pathway. To further study the therapeutic mechanisms and key target of Traditional Chinese Medicine in the therapy of SAP as well. Those studies may provide a theoretical basis for exploitation and development of Dachengqi decoction and a new evidence of Tongli Gongxia method in prevention and cure of acute abdomen and complicated diseases based on the Traditional Chinese Medicine theory of "the internal organs taking pass as use".

重症急性胰腺炎（SAP）是临床危重病，全身炎性反应综合征（SIRS）是SAP发展至多器官功能障碍综合征（MODS）的前期表现，阻断SIRS向MODS的进展具有重要的临床意义。中性粒细胞（PMN）是炎症反应的重要标志，如果其凋亡延迟，将释放大量炎症因子，进而加重SIRS。本课题组在前期研究的基础上，从细胞体外实验和临床研究两方面，基于"六腑以通为用"理论，采用通里攻下法治疗SAP为切入点，以炎症反应时PMN凋亡异常为主线，应用多种细胞分子生物学技术，从PMN的内质网形态、未折叠蛋白反应、钙离子稳态等方面进行内质网应激介导的凋亡途径异常的研究。在更深层次解析重症急性胰腺炎SIRS时PMN凋亡延迟和内质网应激介导的凋亡信号传导途径异常的分子机制及中药的治疗机理和作用靶点。基于祖国医学的"以通为用"理论，为通里攻下法防治急腹症疑难疾病提供新的依据，促进经典验方大承气汤的开发和利用。

重症急性胰腺炎（SAP）是临床常见的急危重症之一，病死率居高不下，尤其在SAP发展过程中，若不及时干预治疗，可导致全身炎性反应综合征（SIRS）和多器官功能障碍综合征（MODS）。中性粒细胞（polymorphonuclear neutrophils，PMN），是炎症反应的重要标志，在炎症的发生、发展和转归中发挥着关键的作用。在SAP的前期阶段PMN对机体起着保护作用，但随着炎症的发展PMN逐渐增多，同时在感染或创伤等应激状态下其生命周期延长。因此，课题组基于“六腑以通为用”的理论从内质网应激介导细胞凋亡的角度对PMN在炎症环境下发生延迟凋亡的分子机制开展深入研究，并探讨大承气汤君药大黄的主要成分大黄素（Emodin）在治疗过程中的作用机制。课题组首先通过SIRS动物模型观察到PMN发生延迟凋亡，然后，比较大黄素和地塞米松对于PMN凋亡的促进作用，并发现大黄素可影响胞内钙稳态，进而促进重症急性胰腺炎SIRS大鼠PMN的凋亡。同时，课题组在细胞和蛋白水平检测到大黄素是通过调控内质网应激通路从而介导PMN细胞发生凋亡，但通路上游的JNK和CHOP并未参与此调控过程。课题组发现大黄素的干预升高了PMN胞内的钙离子水平，因此激活calpain 1，活化的calpain 1识别并剪切了内质网介导凋亡通路的上游凋亡信号因子caspase 12并使之活化，被激活的caspase 12又进一步结合并剪切caspase 3并使之活化，最后诱导PMN的凋亡。中性粒细胞的凋亡能够减少TNF-α，IL-6等细胞因子的释放，进而抑制SAP发展为SIRS而降低了重症急性胰腺炎的死亡率。本项目通过重症急性胰腺炎SIRS动物模型和中性粒细胞凋亡延迟的实验研究，揭示了大黄素通过对内质网应激通路的调控作用，有效地遏制SIRS的发展，避免MODS的发生，从而降低SAP的病死率，提高治愈率。为大承气汤进一步开发和利用奠定理论基础，为祖国医学中的“六腑以通为用”理论及通里攻下法防治重大疑难疾病提供现代化的科学依据。