P2X7R和TLR4信号交互调控NLRP3炎症小体在哮喘气道重塑中的作用及机制

Th1/Th2 imbalance is an important basis of bronchial asthma, and the advantage of Th2 is a key of asthma occurrence and development. P2X7R-mediated NLRP3 inflammasome signaling pathway is considered as a new therapeutic target because of the important role in airway inflammation. Our previous studies revealed a positive correlation when treated P2X7R and TLR4 in BEAS-2B cells with LPS. Antagonist P2X7R could inhibit the activation of LPS/ATP-stimulated airway epithelial cells through NLRP3 inflammasome; PD could relieve airway remodeling by depressing the expression of protein P2X7R and TLR4. Therefore, we proposed that PD could cut off the interaction between macrophages and airway epithelial cells, improving the evolution of airway remodeling, based on cross-talk of the signaling pathways between P2X7R and TLR4. This research intends to investigate the key target cells and key sites of cross-talk of the signaling pathways between P2X7R and TLR4 in asthma airway remodeling using gene overexpression, silencing, RNA interference and co-culture in vitro models as a means of research, elucidating the mechanism of PD mediated cross-talk of signaling pathway between P2X7R and TLR4 to improve airway remodeling. This study provides a new target and theoretical basis for prevention and treatment of asthma airway remodeling and new drug development.

Th1/Th2失衡是支气管哮喘发病的重要基础，Th2优势是哮喘发生发展的关键环节。P2X7R介导的NLRP3炎症小体信号途径在气道炎症中起重要作用，为新的治疗靶点。我们前期研究发现：LPS干预BEAS-2B细胞内P2X7R和TLR4呈正相关，P2X7R拮抗剂通过NLRP3炎症小体抑制LPS/ATP联合刺激的气道上皮细胞活化；且虎杖苷（PD）通过抑制P2X7R与TLR4蛋白的表达，缓解气道重塑。由此提出，PD 基于P2X7R和TLR4信号通路的交叉对话，进而切断巨噬细胞-气道上皮细胞间交互，将改善气道重塑的演进。本课题拟利用基因过表达、沉默和RNA干扰及共培养体外模型等技术，探讨P2X7R与TLR4 信号通路交叉对话在哮喘气道重塑中的关键靶细胞及其关键位点，明确PD介导P2X7R与TLR4信号通路交叉对话改善气道重塑的作用机制。为寻找防治哮喘气道重塑及新药开发提供新的靶标和理论依据。

为阐明虎杖甙(PD)通过TLR4和P2X7R信号交互缓解哮喘气道重塑的免疫调节机制，项目组圆满完成了3项计划研究内容，并进行了拓展研究。建立体内和体外气道重塑模型，通过检测免疫组化、ELISA、Western blot等分子生物学指标，（1）发现：TLR4的敲除或过表达显著影响THP-1细胞的IL-1β释放，并通过NOX/ROS信号通路调节BEAS-2B细胞中的线粒体ROS产生。PD通过调节TLR4/P2X7R和NLRP3炎症小体减弱哮喘小鼠的气道炎症、肺组织ROS水平和凋亡以及IL-1β、IL-18的释放。在LPS+ATP处理的THP-1细胞中，PD显著抑制了TLR4/P2X7R信号通路及其下游IL-1β、IL-18的释放以及钙的内流。PD预处理，减少了TLR4/P2X7R介导的BEAS-2B细胞的线粒体ROS产生和凋亡。干预THP-1细胞TLR4/P2X7R轴会导致哮喘BEAS-2B细胞的线粒体损伤和凋亡。（2）发现：在体外，IL-13对BEAS-2B细胞的刺激导致NOX4的显著上调，伴随着线粒体损伤诱导的凋亡、NLRP3/IL-1β激活和miRNA-182-5p的减少；miRNA-182-5p的过表达显著抑制上皮细胞凋亡和NLRP3/IL-1β的激活。在体内，miRNA-182-5p agomir治疗显著降低了支气管肺泡灌洗液中嗜酸性粒细胞的百分比，并下调了IL-4、IL-5、IL-13等炎症因子。同时，miRNA-182-5p agomir减少了支气管周围炎性细胞浸润、杯状细胞增殖和胶原沉积。（3）发现：在体外miR-181b-5p通过调节BEAS-2B和16HBE细胞中的Wnt配体，抑制DEK/p-GSK-3βSer9/β-catenin/MMP-9通路的活化。在体内小鼠模型中，miR-181b-5p agomir通过DEK对气道炎症和重塑具有抑制作用。miR-181b-5p可以通过抑制NETs释放、DEK/p-GSK-3βSer9/β-catenin/MMP-9途径、DEK/Wnt/DRP1/MMP-9和线粒体损伤来减轻中性粒细胞性哮喘。