百部汤多靶位改善肺泡微环境抑制肺纤维化的作用机制研究

Pulmonary fibrosis is a difficult and severe disease in respiratory diseases. However, haze and cigarette smoke are the main environmental factors that cause the increase of incidence and mortality of pulmonary fibrosis. The mechanism of pulmonary fibrosis is complex. The vicious cycle between “the dysfunctional interaction of alveolar epithelial cells, alveolar macrophages and fibroblasts” and “the imbalance of alveolar microenvironment” is the key to the development of pulmonary fibrosis. Therefore, the effects of the drugs only against one or several cytokines or pathways are often poor. Treatment of pulmonary fibrosis with traditional Chinese medicines has achieved good results, but the mechanisms of most treatments are not clear. Baibutang is one of traditional prescriptions for the treatment of “lung impotent”. Our previous study showed that Baibutang had a significant inhibitory effect on the experimental pulmonary fibrosis, especially the high survival rate of animal (100%), compared with Baibu (roots of Stemona tuberosa, with survival rate of 70%). On the basis of the establishment of a mouse model of PM2.5/cigarette smoke extract + bleomycin and a platform of multi-cell co-culture system, this project aims to study the effects of Baibutang and its three functional groups (Zhike pingchuan group, Yiqi yangyin group and Lishi jianpi group) on the interaction between the three dysregulated key cells and the imbalance alveolar microenvironment, and to clarify the main contributions and main effects of different functional groups in the treatment of pulmonary fibrosis. The results will verify the hypothesis of this project: Targeting the interaction of different cells and the microenvironment of pulmonary fibrosis, Baibutang suppresses pulmonary fibrosis with the integrated power of three functional groups by performing their respective duties and working in cooperation.

肺纤维化是呼吸系统疾病中的疑难重症，而雾霾天气与香烟烟雾是导致肺纤维化发病率和死亡率上升的主要环境因素。肺纤维化机制复杂，肺泡上皮细胞、巨噬细胞和成纤维细胞相互作用失调与肺泡微环境失衡之间的恶性循环是肺纤维化发生发展的关键。中药复方治疗肺纤维化取得了较好的效果。百部汤为治疗肺痿的古方，具有显著的抗肺纤维化和提高动物生存率的作用。本项目拟构建“PM2.5/香烟烟雾提取物+博来霉素”小鼠模型和多细胞共培养体系，在研究百部汤三个功能组（止咳平喘、益气养阴和利湿健脾）对细胞相互作用失调和肺泡微环境失衡影响的基础上，探究不同功能组在治疗肺纤维化中的主要贡献、作用机制和主要效应物质，阐明百部汤通过不同功能组靶向不同细胞/信号通路改善肺泡微环境、“标本兼治”肺纤维化的机制。揭示止咳平喘、益气养阴和利湿健脾类药物靶向的细胞/信号通路，对于肺纤维化及同类疾病的中医临床用药和创新中药研发均具有重要的指导意义。

肺纤维化是呼吸系统疾病中的疑难重症，而雾霾天气与香烟烟雾是导致肺纤维化发病率和死亡率上升的主要环境因素。本课题以治疗肺痿的古方百部汤(BBT)为例，探索了BBT组成药物的协同机制及研究方法。首先制备了PM2.5+博来霉素(BLM)、香烟烟雾提取物(CSE)+BLM的小鼠模型，研究了BBT及三个功能组（止咳清热组ZK、益气养阴组YY和利湿健脾组LS）的干预作用。结果表明，单独滴注PM2.5或CSE主要引起肺部炎症，但PM2.5或CSE与BLM联合滴注则大大加重了BLM引起的肺纤维化，提示PM2.5或CSE对有呼吸系统疾病的患者影响很大；BBT对肺纤维化具有显著的改善作用，而ZK、YY、LS组分别在改善纤维化指标、抑制细胞凋亡、抑制PLA2 G4A/G7亚型方面具有最显著的作用。根据肺痿的特点制备了阴虚肺纤维化模型，检测与纤维化、阴虚和水液代谢等相关的指标研究BBT的干预作用和机制。结果显示，BBT对大多数指标具有很强的改善作用，而ZK组改善肺纤维化和调节肾素-血管紧张素系统平衡的作用最好，YY组改善阴虚指标和cAMP信号通路的作用最好，LS组改善水液代谢和调节肺表面活性蛋白的作用最强且优于BBT，揭示了BBT通过不同功能组靶向不同信号通路协同共治肺纤维化的机制；建立了复方药物协同作用的半定量估算方法，相对药效值RPV用于比较三个功能组在相同剂量下的作用大小，贡献值CV用于比较每个功能组对BBT药效贡献的大小，配伍系数CC为三个功能组的协同作用在BBT药效中的占比。采用LC/MS研究了BBT的物质基础，鉴定了54个主要成分及其对应靶点，其中37个成分与肺纤维化、阴虚和肺水肿等73个靶点相关；分析了肺纤维化大鼠内源性代谢物的变化，BBT对差异代谢物的调控可能涉及到14条代谢通路。研究了老龄大鼠肺纤维化的特点以及BBT的改善作用，采用转录组学比较了老龄和低龄大鼠肺纤维化的差异基因以及BBT可能靶向的潜在基因。肺纤维化机制复杂，多细胞相互作用失调导致肺泡微环境失衡可能是肺纤维化的关键，采用细胞共培养研究了它们之间的关系，结果显示细胞共培养只适合研究细胞间的直接作用，间接作用很难模拟。