基于Notch信号通路及其表观遗传改变探讨养肺活血方防治肺纤维化的作用机制

Pulmonary fibrosis is the result of body's self-repair after lung injury and a common outcome of a variety of lung diseases. PF is a refractory disease which lacks specific drugs in clinic; therefore it has become a hot spot in current medical research. Yangfei Huoxue Prescription is composed of drugs for promoting blood circulation and removing obstruction in channels and drugs for supplementing Qi and nourishing Yin, whose mechanism relates to the interference of signal regulatory factors like the signaling pathway of ERK1/2 MAPK, transforming growth factor β（TGF-β）, tumor necrosis factorα（TNF-α）, nuclear factor kappa B（NF-ｋB）and vascular endothelial growth factor（VEGF）. This project sets the Notch signaling pathway as the starting point, observing the influence that Yangfei Huoxue prescription have on the expression of Notch receptor (Notch1-4), Notch ligands (Jagged1, Jagged2, DLLl, DLL3 and DLL4) and CSL of PF with application of molecular biology techniques such as RT-PCR, Westenblot and flow cytometry. This project also aims to explore the activating function of methylated Notch genes from epigenetic perspective, identifying changes of Furin proteolytic enzyme, metalloprotease and Υ-secretase involved in the activation process .Meanwhile this project studies the connections between Toll-like receptors, costimulatory molecules, Th1/Th2 drift and Notch signaling pathway during the process of pulmonary fibrosis for the purpose of determining the core target sport when treating with Yangfei Huoxue prescription.

肺纤维化（PF）是肺损伤后人体自身修复的结果，也是多种肺部疾病的共同结局。本病为难治性疾病，临床缺乏特效药物，是目前医学研究的热点。养肺活血方由活血通络、益气养阴药组成，作用机制与干预ERK1/2MAPK信号通路和TGF-β、TNF-α、NF-ｋB、VEGF等信号调控因子有关。 本项目围绕与上述密切相关的Notch信号通路为着眼点，采用实时定量PCR、Westem blot、流式细胞术等分子生物学技术，观察养肺活血方对PF模型Notch受体（Notch1－4）、Notch配体（Jaggedl、Jagged2、DLLl、DLL3和DLL4）、CSL表达的影响，并从表观遗传学角度探讨Notch基因甲基化对其活化的作用，明确活化过程Furin蛋白酶、金属蛋白酶、Υ-促分泌酶的变化。同时，研究PF时Toll样受体、共刺激分子、Th1/Th2漂移与Notch信号通路的关系，寻找该方的核心靶点。

肺纤维化是损伤后人体自身修复的结果，也是多种肺部疾病的共同结局。本病为难治性疾病，临床缺乏特效药物，是目前医学研究的热点之一。养肺活血方是临床验方，由活血通络、益气养阴药组成，作用机制与干预ERK1/2 MAPK信号通路和TGF-β、TNF-α、NF-ΚB、VEGF等信号调控因子有关。本项目以Notch信号通路为着眼点，采用Westernblot、PCR、流式细胞术及基因芯片等分子生物学技术，从Notch受体、配体、CSL、基因甲基化及Notch信号活化涉及到的主要蛋白酶水平的变化、Th1/Th2漂移、免疫反应紊乱、微血管新生失控、纤维组织增生等多靶点效应等方面，探讨了养肺活血方防治肺纤维化的作用机制。实验结果表明，养肺活血方通过抑制博来霉素致大鼠肺纤维化模型肺组织及TGF-β1致原代培养乳鼠成纤维细胞增殖模型Notch信号通路相关蛋白的表达，降低鼠肺成纤维细胞的增殖，从而改善肺泡炎及肺间质的纤维化程度。同时，养肺活血方能够抑制单个核细胞Notch信号通路相关蛋白的表达，通过纠正Th1/Th2失衡、抑制TLR4受体活化后间接抑制NF-κB的活化等途径，干预炎性因子的过度表达，减少成纤维细胞增殖及细胞外基质的沉积。此外，该方还通过抑制Notch4及DLL4的表达，使CSL表达减少，进而反馈性抑制VEGF的表达，使肺微血管的异常增生受到阻抑。本项目研究成果，从实验角度证实活血通络与益气养阴配伍组方治疗肺纤维化具有协同增效的良好效果。不仅为肺纤维化的中医临床治疗提供了重要的理论指导和可靠的实验室依据，而且在初步分析复方有效成分的基础上，为进一步的新药开发奠定了良好基础。