PM2.5促进肺纤维化形成的机制及保肺化纤方调控TGF-β和wnt/β-catenin通路的作用

Idiopathic pulmonary fibrosis (IPF) is a severe disease with high rates of disability and mortality, and poor prognosis, without effective therapeutics. Its increasing morbidity has been strongly linked to aggravating air pollution, however, the underlying mechanism remains unclear. Based on the pathogenesis theory coursing fibrosis of vital deficiency, collaterals impediment and impairment accumulation, we aim to use a bleomycin (BLM)-induced IPF rat model and human type II alveolar epithelial cell (A549) and lung fibroblast (MRC-5) models as study objects, and expose the models to air pollutant particulate matter (PM) 2.5. The affecting characters and mechanisms of PM2.5 on the IPF rat model will be determined with pathological, laser scanning confocal microscope, quantitative polymerase chain reaction, immunohistochemical and western blotting methods. Furthermore, we will observe the effects of Baofei Huaxian granules, a traditional Chinese medicine with efficacy of invigorating lung qi, promoting blood circulation and resolving phlegm and dredging collaterals, on pulmonary histomorphology, and productions of hydroxyproline, reactive oxygen species, cytokines, chemokines and extracellular matrix, and the functions of A549 and MRC-5 cells, and TGF-β and Wnt/β-catenin signaling regulation. Through this study, we wish to observe the impact characters of PM2.5 on the BLM-induced IPF rat model, and explore the effective features of Baofei Huaxian granules on regulating epithelial-mesenchymal transition via TGF-β and wnt/β-catenin signaling regulations, and provide scientific basis for searching more beneficial therapeutics.

特发性肺纤维化（IPF）致残率高、死亡率高、预后不良，尚缺乏有效疗法。其日益增高的发病率与大气污染加剧密切相关，但相关机制尚不明确。本项目基于“正虚络痹积损”致纤维化的病机理论，以博来霉素（BLM）诱导的IPF大鼠模型和A549、MRC-5细胞模型为对象，并采用PM2.5暴露，采用病理学、激光共聚焦显微镜、qPCR、免疫组化/印迹等技术，研究PM2.5对BLM诱导的IPF大鼠模型的影响特点和机制；并在此基础上观察具有补肺益气、活血化痰通络功用的保肺化纤方对肺组织形态学和羟脯氨酸、活性氧、细胞因子、趋化因子和细胞外基质及A549、MRC-5细胞功能和TGF-β、Wnt/β-catenin信号通路的影响，揭示保肺化纤方的效应特点及其通过调控TGF-β和wnt/β-catenin信号通路调节上皮间质细胞转化的作用机制，为寻求有效治疗手段提供科学依据。

目的：观察PM2.5暴露对BLM诱导的IPF大鼠模型的影响特点及保肺化纤方调控TGF-β1/Wnt/β-catenin通路的作用机制。方法：（1）实验一大鼠分为对照组、BLM组、PM2.5组和BLM+PM2.5组，实验二分为对照组、BLM+PM2.5组、保肺化纤组、吡非尼酮组、XAV-939组、保肺化纤+吡非尼酮组、保肺化纤+XAV-939组，以BLM气管内雾化制备IPF大鼠模型，采用实时在线浓缩系统进行PM2.5暴露，检测肺功能、肺组织形态学，测定肺组织羟脯氨酸含量、胶原沉积，检测血清、BALF和肺组织细胞因子和细胞外基质及TGF-β1/Wnt/β-catenin通路相关基因和蛋白表达，对实验一四个肺组织进行单细胞测序。（2）以A549和MRC-5细胞为研究对象，分别用BLM、PM2.5及其联合进行刺激，观察细胞迁移、增殖、凋亡、转化及相关细胞因子、趋化因子、ECM及TGF-β1/Wnt/β-catenin相关基因和蛋白表达。结果：（1）PM2.5暴露可显著降IPF大鼠肺功能，诱发外周血和BALF中性粒细胞、淋巴细胞等炎症细胞升高，提高血清和BALF炎症因子水平，加剧肺组织病理损伤和胶原沉积，提高肺组织羟脯氨酸、细胞因子和细胞外基质及TGF-β1/Wnt/β-catenin通路相关基因和蛋白表达。保肺化纤方可以显著改善上述指标，尤以保肺化纤方联合吡非尼酮、XAV-939组改善更为显著。建立了正常、BLM、PM2.5和BLM+PM2.5大鼠肺组织单细胞转录组图谱，揭示PM2.5加剧IPF的转录组特征。（2）PM2.5可显著抑制正常和BLM刺激的A549和MRC-5细胞活性和迁移，诱导细胞凋亡和ROS生成，上调α-SMA表达，促进IL-1β、LN、FN、TGF-β1等分泌，上调MUC5ac、MUC5b、ICAM1、eotaxin-1、MCP-2、MIP-2、TGF-β、wnt1、β-catenin等基因和蛋白表达。保肺化纤方可显著降低上述指标，尤以保肺化纤方联合吡非尼酮、XAV-939组改善更为显著。结论：（1）PM2.5暴露可显著加剧BLM诱导IPF大鼠肺部炎症反应，增加胶原沉积，加剧肺纤维化进程，TGF-β1/Wnt/β-catenin通路可能是其主要机制。（2）保肺化纤方可下调该通路，减轻肺组织炎症反应，对PM2.5引起的上述损伤起到很好的保护作用。