温肺补气通络方调控线粒体功能干预特发性肺间质纤维化分子机制研究

Idiopathic pulmonary fibrosis (IPF) is a fatal disease and there are currently limited options for effective treatment. Mitochondrial dysfunction changes energy metabolism of endothelial cells, fibroblasts, and immune cells, eventually leads to apoptosis and senescence, which is an important pathological feature of IPF. Therefore, the maintenance of mitochondrial homeostasis could be a key strategy for the treatment of IPF. Based on the theory of "Chronic Diseases Transforming to Collaterals" and clinical practices for many years, Wenfei Buqi Tongluo decoction (WBT) was formed on the basis of the addition and subtraction of Buyang Huanwu Tang. Clinical and animal studies found that WBT significantly reduced inflammation and pulmonary fibrosis by improving the symptoms of IPF, such as shortness of breath and expectoration, and decreasing the expressions of TNF-ɑ, TGF-β1 and connective tissue growth factor CTGF. Recent studies found that WBT significantly increased ATP production and mitochondrial respiration; which suggests that WBT has the function of enhancing mitochondrial function, but its molecular mechanism is not yet clear. This project aims to systematically reveal the molecular mechanism of WBT on delaying the progression of IPF by regulating mitochondrial function in terms of inflammatory response, epithelial-mesenchymal cell transformation and fibrosis in bleomycin-induced IPF mouse models and TGF-β1-induced cell models. This project will provide experimental basis of WBT to further understand the treatment of IPF.

特发性肺间质纤维化（IPF）是一种致死性疾病，目前有效治疗选择有限。线粒体功能紊乱改变内皮细胞、成纤维细胞和免疫细胞的能量代谢，最终导致细胞凋亡和衰老，是IPF的重要病理特征。因此，通过调控线粒体功能维持其稳态是IPF治疗的关键。基于“久病入络”理论和多年临床实践，在经方补阳还五汤加减基础上形成温肺补气通络方。临床和动物研究证实，本方明显改善IPF气短、咯痰等症状，并降低TNF-ɑ、TGF-β1和结缔组织生长因子CTGF的表达，提示显著减轻炎症反应和肺纤维化程度。最新研究发现，本方能显著促进ATP产生及线粒体呼吸，确定了其具有增强线粒体功能的作用，但其分子机制尚不清楚。本项目拟在博莱霉素诱导的肺纤维化小鼠模型及TGF-β1诱导的肺纤维化细胞模型上，从炎症反应、上皮-间质细胞转化和纤维化进程方面系统揭示该方通过调控线粒体功能来干预IPF进展的分子机制，为深入理解该方治疗IPF提供实验依据。

特发性肺纤维化(IPF)是一种进展性疾病，死亡率高，预后差。常规治疗的预后仍然有限。温肺补气通络方（WBT）是临床经验方，应用于治疗肺纤维化等呼吸系统疾病。本项目在博莱霉素（BLM）诱导的肺纤维化小鼠模型及TGF-β1诱导的肺纤维化细胞模型上，从炎症反应、上皮-间质细胞转化和纤维化进程方面系统揭示该方通过调控线粒体功能来干预IPF进展的分子机制。项目组利用在体实验，通过Micro-CT小鼠成像、病理组织染色，WB和PCR等实验方法，证实WBT具有抗炎、抗纤维化作用。并在此基础上进行多层次-多靶点机制探索，初步发现WBT方能在组织病理学水平上改善BLM模型小鼠的炎症渗出和胶原沉积。与BLM诱导组比较，WBT方干预后IL-6、IL-1β、TNF-α水平降低；磷酸化NF -κB p65、TLR4、MyD88的表达均显著下调，具有明显抗炎作用。其次，在博莱霉素诱导的小鼠模型和TGF-β1诱导的细胞模型中，WBT抑制TGF-β和Smad3磷酸化水平，进而减轻EMT和ECM积累，具有抗纤维化作用。离体实验中，使用TGF-β1诱导A549及TC-1细胞建立EMT细胞模型，WBT干预后发现，细胞增殖及E-cardherin、N-cardherin、α-SMA以及Collagen I等关键细胞因子的表达分泌有明显变化，提示其对肺泡上皮细胞的EMT过程有抑制作用，同时发挥调控线粒体功能。研究结果提示，WBT可通过抑制TLR4/MyD88/NF-kB通路减弱BLM诱导的炎症反应；通过TGF-β/Smad3途径抑制EMT，促进ECM降解，从而阻断PF的进程，初步揭示了温肺补气通络方对特发性肺纤维化相关机制，为中药复方抗炎、抗纤维化临床应用提供了实验依据。