灯盏花乙素对NLRP3炎症小体活化和细胞焦亡的抑制作用及其机制研究

The NLRP3 inflammasome is an important component of the innate immunity, whereas the aberrant activation of NLRP3 inflammasome closely correlates with many inflammatory diseases including infections, injuries, and metabolic disorders. It is thus likely that the inhibitors of NLRP3 inflammasome activation are promising therapeutic agents for such inflammatory diseases. The traditional Chinese medicine Erigeron breviscapus and its extract Breviscapine possess significant anti-inflammatory activities and have been used to treat inflammation and damage-related diseases effectively, but their pharmacological mechanisms remain only partly understood. In our preliminary studies, we have found that scutellarin, the major ingredient of Breviscapine, could efficiently suppress the activation of NLRP3 inflammasome and pyroptosis, but the inhibitory action mechanism is unclear. In the present project, we will explore the effects of scutellarin on the regulatory signaling (including the PKA, Syk, JNK and AMPK pathways) and proteins (such as NEK7 and PKR) for NLRP3 and ASC, thereby identifying the major regulatory signaling in mediating the inhibitory effect of this drug and further elucidating its action mechanism for inhibiting NLRP3 activation and pyroptosis. Moreover, the in vivo inhibitory activity and pharmacological effect of scutellarin will be evaluated in animal models of NLRP3 inflammasome activation and/or bacterial sepsis. The studies of this project will provide the scientific basis for those traditional Chinese medicines or effective ingredients containing scutellarin to treat inflammatory diseases related with aberrant activation of NLRP3 inflammasome.

NLRP3炎症小体是机体固有免疫的重要组成部分，其异常活化与感染、损伤及代谢失调等炎症性疾病密切相关，故NLRP3活化的抑制剂有望成为炎症性疾病的新型治疗药物。中药灯盏花及其提取制剂灯盏花素具有很强的抗炎作用，对炎症、损伤相关疾病有明显疗效，但其药理作用机制仅部分了解。我们在前期研究中发现，灯盏花素中的有效成分灯盏花乙素能明显抑制NLRP3炎症小体的活化和细胞焦亡，但其抑制作用机制不明。本项目拟深入探讨灯盏花乙素对NLRP3、ASC等关键炎症小体组分的调控信号（PKA、Syk、JNK、AMPK等）和调控蛋白（NEK7、PKR等）的影响，确定介导该药物抑制NLRP3活化的主要调控信号分子，阐明其抑制NLRP3活化和细胞焦亡的作用机制，并在体内NLRP3炎症小体活化模型和脓毒症动物模型中印证其药理效应，为含有灯盏花乙素的中药及其有效成分临床应用于NLRP3异常活化相关疾病的治疗提供科学依据。

NLRP3炎症小体不仅是机体固有免疫系统的重要组成部分，而且其异常活化与感染、损伤及代谢失调等炎症性疾病密切相关，因此NLRP3炎症小体活化抑制剂有望成为相关炎症性疾病的新型治疗药物。中药灯盏花及其提取制剂灯盏花素的主要有效成分是灯盏花乙素，具有很强的抗炎作用，对炎症、损伤相关疾病有明显疗效，但其药理作用机制并未完全了解。本项目主要研究灯盏花乙素对巨噬细胞中经典和非经典NLRP3炎症小体活化的潜在抑制作用，从细胞和分子水平阐明其作用机制，并在NLRP3炎症小体活化相关的细菌性脓毒症小鼠模型中验证其体内作用及机制。结果表明，灯盏花乙素不仅能剂量依赖性地抑制激活剂ATP或尼日利亚菌素激活的经典NLRP3炎症小体活化及细胞焦亡，而且能强烈地抑制细胞内脂多糖（LPS）诱导的半胱天冬酶-11活化，进而抑制后者介导的非经典NLRP3炎症小体的组装和焦亡。机制研究揭示，灯盏花乙素能够激活蛋白激酶A（PKA），后者进而对NLRP3或Caspase-11上的PKA特异性位点进行磷酸化修饰，从而抑制NLRP3和Caspase-11活化及相应炎症小体的组装，并有效抑制半胱天冬酶1活化、白细胞介素1β（IL-1β）成熟、GSDMD切割以及细胞焦亡，从而降低成熟IL-1β等炎症因子的释放水平和炎症反应的强度。小鼠细菌性脓毒症模型实验证明，灯盏花乙素能够降低模型小鼠血清中IL-1β的水平和肝脏的炎症损伤程度，显著延长腹腔细菌感染小鼠的存活时间，提高存活比例。这些结果揭示了灯盏花乙素通过抑制NLRP3和半胱天冬酶11炎症小体活化而发挥抗炎作用的新药理机制，同时初步显示了该药物对于细菌性脓毒症等临床重大疾病的防治作用，为将灯盏花中药及灯盏花素制剂用于防治NLRP3炎症小体过活化相关的疾病提供了实验依据。