急性胰腺炎“热-炎”病机的中性粒细胞凋亡分子开关机制及验证

Abstract: In traditional Chinese medicine theory, ‘heat’ evil was a consistent pathogen along with the whole pathological process of acute pancreatitis (AP). It corresponds to the role of systemic inflammatory response in acute pancreatitis under western medicine theory. So it’s believed that ‘heat’ is equated with inflammation. It has been reported that neutrophil apoptosis rate positively correlated with the outcomes of inflammation. Activated C Kinase 1 (RACK1) can suppress Bcl-2 and then promote apoptosis. It has been regarded as a key molecule to regulate apoptosis. Our previous studies have confirmed that RACK1 in leukocytes of AP was negatively correlated with inflammatory mediators. And purgative herbs mixture such as Chaiqinchengqi decoction (CQCQD), which is effective clinically to treat AP, could promote the release of mitochondrial cytochrome C. Moreover, we also have proven that CQCQD could up-regulate the expression RACK1 in alveolar macrophages of AP. Whereby, we proposed that neutrophil apoptosis regulated by RACK1 may be a key in the ‘heat-inflammation’ pathogenesis of AP and also an important target of CQCQD with the effect of ‘Purging fu-viscera to eliminate heat’. In the present study, we intend to investigate the expression of apoptosis-related mRNA and proteins, RACK1 and Bcl-2 family, in peritoneal neutrophil of acute necrotizing pancreatitis rats and verify the effect of CQCQD on mitochondrial permeability transition pore. This study is expected to further elucidate ‘heat’ pathogenesis of AP with modern theory and uncover innovative mechanism of CQCQD to purge fu-viscera to eliminate heat in treating AP.

中医“热”邪贯穿急性胰腺炎（AP）病机的演变始终，与西医全身炎症反应的病理生理作用不谋而合，“热”即是“炎”。中性粒细胞凋亡预示炎症反应的转归；蛋白激酶C受体1（RACK1）可抑制B淋巴细胞瘤-2（Bcl-2），促进细胞凋亡，被认为是调控细胞凋亡的开关蛋白。我们研究发现：白细胞RACK1与炎性介质水平负相关；AP中药验方柴芩承气汤（CQCQD）“通腑泄热”，促进胰腺腺泡细胞线粒体释放细胞色素C，上调巨噬细胞RACK1表达。据此，我们提出假说：RACK1调控中性粒细胞凋亡可能是AP“热-炎”病机的分子开关，且是CQCQD“通腑泄热”治疗作用靶点。本项目检测急性坏死性胰腺炎大鼠中性粒细胞凋亡调控相关蛋白RACK1及Blc-2家族等的基因和蛋白表达变化，验证CQCQD增强中性粒细胞线粒体通透性转换孔的开启效应。有望进一步提升AP“热”邪病机的现代认识，阐明CQCQD通腑泻热作用的创新机制。

中性粒细胞（PMN）参与急性胰腺炎（AP）的局部及全身性并发症，是AP病情进展的关键因素之一。适时的PMN自发性凋亡是机体固有的细胞死亡机制，调节PMN的潜在毒性。中性粒细胞胞外诱捕网（NETs）释放是不同于凋亡的另一种细胞死亡方式，是AP病情发展的重要组成部。本研究以AP大鼠模型，探索柴芩承气汤（CQCQD）调节PMN凋亡的作用机制；以AP患者PMN为研究对象，证实PMN延迟凋亡对AP病情严重程度的影响，并进一步探索PMN延迟凋亡与NETs的关系及CQCQD的作用。动物模型研究结果显示CQCQD调控PMN的RACK1、Bcl2家族蛋白及细胞色素C，促进PMN凋亡，减轻ANP的器官损伤及炎症反应。临床研究显示AP患者血液NETs高表达与PMA诱导新鲜分离PMN的NETs低反应性相矛盾，但进一步研究发现AP患者延迟凋亡PMN具有PMA诱导NETs的高反应性，结合AP患者显著的PMN延迟凋亡，提示PMN延迟凋亡促进AP NETs生成。3MA抑制自噬减少NETs生成，而Rapamycin促进自噬增高NETs生成，进一步为自噬参与PMA诱导NETs生成提供证据。3MA和CQ抑制自噬可促进HV受试者PMN凋亡，并抑制SAP血清诱导的 PMN延迟凋亡，但二者对AP患者PMN凋亡无显著影响。进一步电镜及蛋白检测证实AP患者PMN自噬流异常可能是PMN延迟凋亡的关键。体外研究证实CQCQD可抑制自噬、ROS和Akt磷酸化，减少PMA诱导的NETs生成，同时体内实验证实CQCQD可减少NETs生成减轻AP。本研究进一步深化PMN延迟凋亡对AP病情严重程度的影响认识，阐释CQCQD救治AP的作用机制。