组蛋白乙酰化调节ORMDL3激活NLRP3炎症小体在RSV感染诱发哮喘中的机制研究

RSV is the most common virus causing severe respiratory disease in children under 5 years. RSV infection in early life stage plays an important role in the development of asthma. Current therapy control the episode of asthma induced by virus face to challenge because β2 receptor agonist insensitive or hormone resistance. It is urgent to explore the mechanism of RSV induced asthma to find a new treatment breakthroughs. Our previous study found that the expression of asthma susceptibility gene ORMDL3 increased in the lung tissue of RSV induced asthma animal model, which indicated that ORMDL3 maybe the link between RSV and asthma. RSV infection could activate NLRP3 inflammasomes and cause inflammation cascade effect, and the expression of NLRP3 increased after transfection of ORMDL3 overexpression plasmid to bronchial epithelial cells. The histone acetylase p300 on the promoter of the ORMDL3 gene is the key of its regulatory mechanism. We speculate that histone acetylation regulate the expression of ORMDL3 activates the over release of NLRP3 inflammasome in RSV induced asthma. The aim of this study is to prove the effect of ORMDL3 on the activation of NLRP3 inflammasome through in vivo and in vitro experiments, and to detect the relationship between histone acetylation, ORMDL3 and NLRP3 inflammasome in RSV induced asthma mouse model. The p300 inhibitor C646 can inhibit the expression of ORMDL3 and NLRP3 inflammasome and relieve the pathological changes of asthma. In this study, we will find new intervention and therapeutic targets from the pathogenesis of RSV induced asthma.

RSV是造成5岁以下儿童严重呼吸系统疾病最常见的病毒，婴幼儿RSV感染对哮喘发生发展起重要作用。目前药物治疗在病毒诱发哮喘方面存在困难并出现耐药。我们前期研究发现哮喘易感基因ORMDL3在RSV诱发哮喘动物模型肺组织中表达增高，可能是RSV与哮喘关系间的桥梁。RSV可激活NLRP3炎症体导致炎症扩散，而ORMDL3过表达质粒转染支气管上皮细胞后NLRP3表达也增多。ORMDL3基因启动子上组蛋白乙酰化酶p300与之结合并调控表达。我们推测RSV感染诱发哮喘中，通过组蛋白乙酰化影响ORMDL3表达从而激活NLRP3炎症体过度释放。本课题拟通过体内外实验证明ORMDL3对NLRP3炎症体激活的影响，并在RSV诱发哮喘小鼠模型检测组蛋白乙酰化、ORMDL3与NLRP3炎症体间关系，予p300抑制剂C646可抑制其表达缓解哮喘病理改变。本研究从RSV诱发哮喘的发病机制找出新干预环节和治疗靶点。

呼吸道合胞病毒（RSV）是造成5岁以下儿童呼吸道感染最常见的病毒，婴幼儿RSV感染促进哮喘发生发展。哮喘易感基因ORMDL3可能是婴儿早期反复RSV感染与哮喘关系间的桥梁。糖皮质激素是哮喘治疗首选药物，但病毒诱发哮喘时过度释放NLRP3炎症小体导致激素不敏感且需加大激素量以控制喘息，这会导致免疫功能抑制及炎症扩散。本研究从本文拟从ORMDL3的作用及调控机制出发，证明在ORMDL3在RSV感染对NLRP3炎症小体的影响，组蛋白乙酰化酶p300是否可调控ORMDL3作用。并在生命早期反复感染RSV哮喘模型中探讨ORMDL3、气道炎症、NLRP3炎症小体、组蛋白乙酰化表达情况，p300抑制剂C646是否可通过调节组蛋白化酶抑制ORMDL3达到缓解气道炎症的作用，寻找新的治疗方向。结果：1.支气管上皮细胞ORMDL3过表达导致NLRP3炎症小体的释放2. 组蛋白乙酰化酶P300的组蛋白乙酰化功能是ORMDL3启动子激活所必须的；3.RSV感染支气管上皮细胞后，可导致ORMDL3、NLRP3炎症小体增多及过组蛋白乙酰化状态；ORMDL3siRNA干预可缓解NLRP3的过度释放； C646抑制ORMDL3缓解 NLRP3炎症小体释放；4. 经鼻腔滴注ORMDL3-AAV6制作肺组织过表达ORMDL3小鼠，并检测到NLRP3炎症小体释放; 5. 在生命早期反复感染RSV哮喘小鼠模型建立，其气道炎症和气道高反应性ORMDL3表达增多、肺组织组蛋白乙酰化、NLRP3炎症小体较哮喘组明显增加； 6. P300可调控ORMDL3表达，并且缓解生命早期反复感染RSV哮喘小鼠肺组织NLRP3炎症小体过度激活。结论： RSV 激活ORMDL3导致NLRP3炎症小体释放的，组蛋白乙酰化酶可抑制ORMDL3 表达，缓解生命早期反复感染RSV哮喘小鼠气道炎症、气道高反应性及NLRP3炎症小体释放。