探索天然药物单体墨旱莲抑制巨噬细胞焦亡进而改善支气管肺发育不良病理的机制

Bronchopulmonary dysplasia (BPD) is characterized by premature alveolar developmental arrest. Antenatal exposure to inflammation inhibits lung morphogenesis, thus increasing the risk of developing BPD. Pyroptosis is critical innate immune responses via caspase-1-dependent processing and secretion of interleukin 1β (IL–1β), which in turn induces the expression of many other pro-inflammatory cytokines and exaggerates inflammation. Our previous studies have demonstrated natural drug Eclipta prostrata L. improves alveolar development in antenatal LPS-treated rats. We have also shown that Eclipta prostrata L. inhibits LPS-induced caspase-1 activation and decreases IL–1β release in raw264.7 cells. We further identify pyrin may be a critical target of Eclipta prostrata L using the RNA-sequence technology screens. Pyrin has emerged as key regulator of inflammasomes. We thus examine the involvement of pyrin in the inhibition of pyroptosis by Eclipta prostrata L. In this study, we attempt to address whether Eclipta prostrata L. inhibits the pyroptosis through pyrin, and leads to improve alveolar development. Furthermore, we investigate whether Eclipta prostrata L. influences the interaction of pyrin with ASC or caspase-1, and hence regulates caspase-1 activation and consequently IL–1β production. In summary, our results establish a new molecular mechanism pertaining to disrupted lung morphogenesis following intrauterine inflammation, and may provide a new therapeutic drug for BPD treatment.

胎儿未成熟肺暴露于宫内炎性环境中，是导致早产儿发生支气管肺发育不良(BPD)的重要危险因素，解析BPD炎症反应机制是研究其病理以及治疗的关键点。细胞焦亡是一种新的细胞程序性死亡方式，依赖于Caspase-1并伴有IL-1ß等炎症因子的释放，过度细胞焦亡会诱发多种自身炎症性疾病。我们前期发现，天然药物单体墨旱莲不仅显著改善BPD疾病模型的病理，而且抑制LPS诱导的细胞焦亡。进一步用全转录组测序技术，在LPS诱导的巨噬细胞培养模型中，筛选到一个墨旱莲抑制细胞焦亡的可能作用靶点—Pyrin蛋白。在此基础上，本课题综合应用CRISPR/Cas9基因编辑、基因过表达、shRNA和免疫共沉淀等手段，多角度阐明墨旱莲通过Pyrin抑制细胞焦亡，从而改善BPD的病理机制，明确墨旱莲是否改变了Pyrin结构域的结合能力，并试图明晰Pyrin在细胞焦亡中扮演的角色。本课题的实施将为BPD的防治提供一种新策略。

支气管肺发育不良（bronchopulmonary dysplasia, BPD）的疾病表现主要为肺泡化发育阻滞，肺泡结构简单化。胎儿期未成熟肺暴露于炎性环境中，是BPD的重要致病因素。但BPD确切的致病机制并不明确；此外，在临床上如何改善BPD也是较为关注的重点。以下本研究的主要发现：.1.在羊膜腔注射脂多糖（LPS）建立支气管肺发育不良（BPD）经典的动物模型中，发现墨旱莲EAP20-2可通过抑制IL-1β的分泌改善BPD新生儿鼠的肺泡化阻滞的病理改变。明确EAP20-2可通过调节pyrin蛋白的表达影响炎症小体的组装，从而抑制LPS诱导的IL-1β释放，并从pyrin与ASC的结合角度，发现EAP20-2可以减少LPS诱导的pyrin蛋白和ASC蛋白的结合。.2．并发现CCR5异常表达是BPD发生的重要机制之一。在BPD的模型中发现LPS可诱导CCR5及其配体表达显著增高，并引起IL-1β表达增高，从而导致肺泡化阻滞，与此同时，通过CCR5和IL-1β受体拮抗剂可抑制炎症改善BPD新生鼠肺泡简单化。CCR5可通过增加巨噬细胞在肺里迁移及渗透的能力，进而促进IL-1β的表达。而LI-1β通过抑制LOX酶活性，引起弹性蛋白异常沉积。在BPD患儿外周血也检测到CCR5及其配体水平升高。鲨肝醇（BTA）也可通过抑制CCR5的功能和IL-1β的产生从而改善BPD新生鼠病理改变。此外，还证明LPS可以通过RIP3/NF-kB引起CCR5表达增多是更深入的分子机制。