通塞颗粒/补肺益肾颗粒序贯治疗通过lncRNAs ANRIL调控TLR4/NF-κb信号通路改善AECOPD肺巨噬细胞损伤的机制研究

Acute exacerbation of COPD leads to progressive decline in pulmonary function and quality of life, and lung macrophage injury is an important manifestation of this course. LncRNAs ANRIL can negatively regulate the TLR4/NF- κb signaling pathway to inhibit the inflammatory response and participate in lung macrophage injury. Previous studies have shown that sequential treatment of Tongsai Granules/Bufei Yishen granules in AE period and risk window period (RW) can reduce the number of acute exacerbations and improve the quality of life of patients, but the mechanism has not been elucidated. This study was designed to: (1) repeated acute exacerbations of COPD ratsmodel, the phagocytic function of pulmonary macrophages was observed by flow cytometry, and the expressions of inflammatory cytokines, lncRNAs ANRIL and TLR4/ nf-kb were detected by ELISA, qPCR and western blot. (2) LPS repeated stimulation of human monocytes (THP-1) cell model, the RNAi method to sink lncRNAs ANRIL, neutral red colorimetry, flow cytometry, qPCR, western blot observation of cell phagocytosis, TLR4/NF- b and inflammatory factor expression. To explore the mechanism of sequential treatment of aecopd-rw on pulmonary macrophage injury, and to provide a new strategy for reducing acute exacerbations.

COPD急性加重导致患者肺功能、生存质量进行性下降，肺巨噬细胞损伤是该病程中的重要表现，lncRNA ANRIL可通过负调节巨噬细胞TLR4/NF-κb信号通路抑制炎症应答，参与肺巨噬细胞损伤。前期研究显示通塞颗粒/补肺益肾颗粒序贯治疗AE期及危险窗期（RW）可减少患者急性加重次数，改善生存质量，但作用机制尚未阐明。本研究拟于：①COPD反复急性加重大鼠模型，流式细胞术观察肺巨噬细胞吞噬功能，ELISA、qPCR、western blot法检测炎症因子、lncRNA ANRIL及TLR4/NF-κb表达；②LPS重复刺激人单核细胞（THP-1）细胞模型，RNAi法沉没lncRNA ANRIL，中性红比色法、流式细胞术、qPCR、western blot法观察细胞吞噬功能、TLR4/NF-κb及炎症因子表达。探讨序贯治疗AECOPD-RW对肺巨噬细胞损伤的作用机制，为减少急性加重提供新策略。

1.本研究结果显示，TSG治疗AECOPD的关键活性成分为槲皮素、芹菜素、木犀草素等，主其核心成分TSG4经动物实验及体外实验验证可显著降低大鼠气道炎症反应和平滑肌增生，降低动物血清TNF、IL-17细胞因子水平，显著下调P53、P-P65、P-MAPK1/3、P-AKT蛋白表达。.2. TSG对AECOPD大鼠具有改善肺功能、抑制炎症的作用。TSG4（核心成分）可通过调节MAPK-sirt1-NF-κB通路显著抑制AECOPD肺组织和CSE/LPS诱导的气道上皮细胞衰老。同时，通塞颗粒可通过调节EGFR/ERK信号通路改善慢性阻塞性肺疾病急性加重期上皮屏障损伤，具体表现为在体内/体外实验中，TSG可通过上调ZO-1、E-CA水平，改善细胞通透性，降低p-ERK/ERK1/2表达，从而改善上皮损伤。.3.通塞颗粒序贯补肺益肾颗粒治疗COPD-AECOPD大鼠可以改善肺功能、减轻肺组织损伤，下调TLR4/NF-κb信号通路表达，减轻肺巨噬细胞损伤，从而降低再次急性加重对机体的损伤，其作用机制与调控lncRNAs ANRIL有关；.4.通塞颗粒序贯补肺益肾颗粒联合莫西沙星干预THP-1炎症模型可有效减轻模型的炎症反应，其中沉没lncRNAs ANRIL后，TLR4/NF-κb信号通路表达收到抑制，提示中西医结合序贯治疗方案可减轻炎症反应及肺巨噬细胞损伤，其作用与调控lncRNAs ANRIL降低TLR4/NF-κb信号传导有关。