NLRP3炎性体介导IL-1β通路在慢性阻塞性肺疾病中的激活和作用研究

Chronic obstructive pulmonary disease (COPD) is characterised by persistent inflammation in the lungs and circulation, and the inflammation is further aggravated during acute exacerbations. Interleukin (IL)-1β is a pivotal pro-inflammatory mediator in COPD. We have previously demonstrated that the level of IL-1β and IL-1β pathway activation in the airway are associated with clinical prognosis in COPD, however it remains unclear whether the upstream pathway of IL-1β is activated in COPD, and what is the effect of the upstream pathway. NLRP3 inflammasome is the upstream signalling molecule of IL-1β production and secretion, therefore we hypothesise that NLRP3 inflammasome induced IL-1β pathway is activated in stable COPD, and the level of the pathway activation is further elevated in COPD exacerbation. We will examine the level of sputum genes and proteins expression that involved in the NLRP3 inflammasome induced IL-1β pathway between patients with stable COPD vs healthy controls, and between patients with stable COPD vs COPD exacerbation by using sputum induction technique. In vitro studies of monocyte/macrophage NLRP3 inflammasome induced IL-1β pathway activation and pathway inhibition using siRNA will be performed to explore the role of this pathway in COPD exacerbation. This study examine the role of the important signalling molecule — NLRP3 inflammasome in COPD, and this will identify its effect in COPD pathogenesis and provide target and evidence of anti-IL-1β therapy for COPD.

慢性阻塞性肺疾病（chronic obstructive pulmonary disease, COPD）以肺部和全身炎症为首要特征，且炎症反应在急性加重期进一步加剧。IL-1β是COPD中的关键促炎性因子，我们首次发现IL-1β水平和下游通路的激活与患者预后密切相关。然而，COPD中IL-1β上游通路是否激活以及通路的作用不清。NLRP3炎性体是介导IL-1β形成和分泌的上游信号分子，我们推测NLRP3炎性体介导IL-1β通路在COPD稳定期表达升高，并在急性加重期进一步激活。本课题拟利用诱导痰技术，比较COPD稳定期患者和健康人、稳定期和急性加重期患者痰液NLRP3炎性体介导IL-1β通路基因和蛋白水平；体外对患者外周血单核细胞和诱导痰巨噬细胞进行通路激活实验和 siRNA通路阻断实验，探讨该通路在COPD急性加重中的作用。本课题通过对COPD中IL-1β上游炎症通路关键信号NLRP3炎性体的研究，明确IL-1β在COPD发生机制中的作用，为特异性抗炎治疗提供靶点和科学依据。

COPD以慢性气道炎症为首要特征，且炎症反应在急性加重期进一步加剧。IL-1β水平在COPD患者中升高并参与疾病的病理生理过程，但IL-1β上游通路及确切机制尚不清楚。NLRP3炎性体介导IL-1β通路可能是COPD患者IL-1β上调的上游机制。为验证这一假说，本课题利用诱导痰技术，比较COPD稳定期患者和健康人、稳定期和急性加重期患者痰液和外周血NLRP3炎性体介导IL-1β通路基因和蛋白水平；体外比较COPD稳定期患者和健康人、COPD患者在稳定期和急性加重期时巨噬细胞NLRP3通路水平；利用THP-1单核细胞系分化的巨噬细胞模拟LPS+ATP引起的通路激活，验证NLRP3特异性siRNA和穿心莲内酯对通路的抑制作用；并在COPD患者巨噬细胞进行通路激活和干预实验，以证明NLRP3通路在COPD中的确切作用。研究结果表明，与健康受试者相比，COPD患者肺部NLRP3通路相关基因NLRP3、CASP1、IL1B表达上调，终产物IL-1β水平升高，且在患者出现急性加重时进一步升高。外周血NLRP3通路在稳定期COPD患者 VS 健康对照、患者稳定期 VS 急性加重期比较无差异，说明NLRP3通路的上调局限在COPD患者的肺部，是局部的炎症和免疫反应，不伴有以外周血为代表的全身NLRP3炎症通路的激活。我们在THP-1细胞系分化的巨噬细胞上实现了LPS+ATP对NLRP3通路的激活，验证了NLRP3特异性siRNA对通路的干预抑制作用，转染后通路相关基因和蛋白（NLRP3、Caspase-1、IL-1β）水平下降，并伴有通路终产物IL-1β的降低；此外，完成课题计划之外，我们延伸进行了天然药物成分穿心莲内酯的此通路干预实验，证明穿心莲内酯可抑制NLRP3通路相关基因和蛋白水平。COPD患者巨噬细胞NLRP3通路的体外激活亦可被穿心莲内酯抑制下调。本课题明确了IL-1β在COPD发生机制中的作用，小分子药物NLRP3特异性siRNA和天然药物成分穿心莲内酯可通过抑制NLRP3通路降低IL-1β水平，为COPD提供了新的抗炎治疗选择和科学依据。