补肺益肾方调控肺成纤维细胞NF-κB通路改善COPD气道重塑机制研究

Airway remodeling, one of the most important pathogenesis of continuous progressive airflow limitation in chronic obstructive pulmonary disease (COPD), and the abnormality of lung fibroblasts (LFs) plays an important role in the pathologic processes. Depressing or reversing the course of airway remodeling will fundamentally relieve the decline of pulmonary function. Our previously completed study indicates that Bu-Fei Yi-Shen granule can significantly improve the pulmonary function, but whether the mechanisms involving in reversing the airway remodeling or not, which needs more further study. In this protocol, we intended to explore the molecular mechanisms of Bu-Fei Yi-Shen Granule via regulation of NF-κB signaling in LFs in COPD, based on ① COPD model rats, we will measure the levels of serum cytokines and extracellular matrix (ECM) by enzyme-linked immunosorbent assay (ELISA), assess the proliferation, apoptosis, migration of LFs by flow cytometric (FCM) and Boyden chamber technologies, and cytokines, ECM and NF-κB pathway relevant factors in lung tissues measured by quantitative real time PCR (qPCR) and western blotting, and also the mentioned parameters in cultured LFs isolated from lung tissues; ② cigarette smoke extract- and/or lipopolysaccharide- stimulated human LFs (HLFs), we will observe the proliferation, apoptosis, migration and production of cytokines and ECM in HLFs, and NF-κB pathway relevant factors, by using serum pharmacological, small interfering RNA, Boyden chamber, flow cytometric, ELISA, qPCR and western bloting technologies. In this study, we purpose to investigate the mechanisms of Bu-Fei Yi-Shen granule reversing the airway remodeling progress, and provide a study basis for clarification of the mechanisms of Chinese medicine in airway remodeling in COPD, and provide a new target point for COPD prevention and treatment.

气道重塑作为慢性阻塞性肺疾病（COPD）气流受限持续进展的重要病理基础，肺成纤维细胞(LFs)功能失常是其关键机制之一。阻抑气道重塑将可能从根本上缓解肺功能持续下降。前期研究表明，补肺益肾方可显著改善COPD患者肺功能，其机制是否涉及减轻气道重塑，尚需深入研究。本研究拟基于①COPD大鼠模型：采用ELISA法观察血清中细胞因子和细胞外基质（ECM），流式细胞术观察LFs增殖、凋亡、迁移及转化，定量PCR（qPCR）和Western blot观察肺组织细胞因子、ECM及核因子（NF）-κB、IκBα和LFs中上述指标表达；②香烟烟雾提取物（CSE）/脂多糖（LPS）刺激的人肺成纤维细胞（HLFs）：采用血清药理学、siRNA、Boyden chamber、流式细胞术、ELISA、qPCR和Western blot等，观察LFs增殖、凋亡、迁移、转化和细胞因子、ECM生成及NF-κB通路变化。探讨补肺益肾方调控LFs NF-κB通路治疗COPD气道重塑机制，为其防治提供新靶点。

背景：COPD是严重危害公众健康的重大疾病，气道重塑是其气流阻塞和疾病进展的主要病理基础，目前尚无特效疗法。补肺益肾方可稳定期患者临床症状，提高运动能力和生活质量等，疗效可靠。本研究基于香烟烟雾联合细菌感染暴露的COPD大鼠模型和CSE、LPS刺激MRC-5细胞模型，观察补肺益肾方对大鼠肺功能、气道壁厚度、血清和肺组织细胞因子、细胞外基质及相关胶原蛋白酶的表达以及MRC-5细胞功能的影响，探讨其作用机制。.方法：（1）SD大鼠随机分为对照组、模型组、补肺益肾方高、中、低剂量组、PDTC组、中剂量+PDTC组、氨茶碱组。第9-20周分别以相应的药物灌胃后，检测肺功能，取血清、肺组织和原代培养的LFs检测细胞因子、细胞外基质、胶原酶及NF-κB信号通路等相关基因和蛋白表达。（2）分别采用补肺益肾方和氨茶碱含药血清及其联合NF-κB siRNA对CSE和LPS诱导的MRC-5细胞进行干预，观察其增殖、凋亡、迁移、转化，以及相关细胞因子、细胞外基质、胶原酶及NF-κB信号通路等相关基因和蛋白表达。.结果：（1）COPD大鼠肺功能较对照组下降明显（P<0.01），上述检测的血清、肺组织和原代培养的LFs细胞的细胞因子、细胞外基质、胶原酶及p-NF-κB p65、p-IκBα等均显著升高（P<0.01）。与模型组比较，各治疗组上述指标均不同程度地降低，其中以补肺益肾方中剂量+PDTC组、高剂量组和中剂量组疗效尤为突出，优于低剂量组、PDTC组和氨茶碱组。（2）补肺益肾方和氨茶碱含药血清可提高siRNA和非siRNA 干预MRC-5细胞增殖率（P<0.01），降低凋亡率（P<0.01），提高上述细胞因子表达（P<0.01），但对细胞外基质、胶原酶及NF-κB无显著影响。CSE和LPS均可显著提高siRNA和非siRNA 干预MRC-5上述指标表达（P<0.01），补肺益肾方和氨茶碱血清及其与NF-κB siRNA联合可降低上述指标（P<0.05），其中以补肺益肾方+NF-κB siRNA疗效尤为突出，优于氨茶碱+NF-κB siRNA组和补肺益肾方和氨茶碱含药血清单独干预组。.结论：补肺益肾方可以降低COPD肺部、系统和CSE/LPS刺激的MRC-5细胞炎症反应，减少细胞外基质和胶原蛋白酶分泌，抑制NF-κB通路相关基因和蛋白表达，减轻气道壁增厚程度，从而发挥抗气道重塑作用。