芸香科生物碱通过干预COX2/PGE2介导的EMT预防ARDS相关肺纤维化的机制研究

Acute respiratory distress syndrome (ARDS) associated pulmonary fibrosis is a high mortality disease characterized by diffuse damage of lung epithelial cells and excessive repair. Epithelia-mesenchymal transition (EMT) is an essential part of pulmonary fibrosis. Previous studies showed that the rutaceae alkaloids potently inhibited pulmonary fibrosis, and unregulated the levels of E-cadherin, COX2/PGE2, and reduced the levels of SMA. These results suggested that the anti-fibrotic effect of the alkaloids might be correlated with COX2/PGE2-mediated EMT. Therefore, we creatively suggest that rutaceae alkaloids might inhibit EMT that would be the new mechanism of anti-pulmonary fibrosis. The current project focuses on clarifying the correlation between the antifibrotic effects and EMT of lung epithelial cells of rutaceae alkaloids using ARDS-associated pulmonary fibrosis model. Then, using the techniques of gene transfection, gene knockout, and molecular biology, the project aims at investigation on the effects of the alkaloids on EMT of epithelial cells, COX2/PGE2 and downstream signaling molecules. Furthermore, the project takes aim at clarifying the mechanism of the alkaloids on regulation of epithelial EMT via COX2/PGE2 signal pathways. The project may elucidate the anti-ARDS associated pulmonary fibrosis effect and mechanism of the rutaceae alkaloids from a novel perspective, and provide a new insight on developing antifibrotic agent for clinical application.

肺上皮细胞广泛受损及过度修复，是急性呼吸窘迫综合症（ARDS）相关肺纤维化的特点，也是高死亡率的原因之一。上皮-间质转化（EMT）是肺纤维化的重要环节。研究提示芸香科生物碱具有抗肺纤维化作用，增加肺上皮细胞E-cadherin，减少SMA，且上调COX2/PGE2，提示可能与调控COX2/PGE2介导的EMT相关。为此，本研究创新性提出调控肺上皮细胞EMT可能是芸香科生物碱抗纤维化的机制。因此，本项目以ARDS相关肺纤维化体内外模型，研究芸香科生物碱抗纤维化作用与抑制EMT的关系；然后采用基因转染、基因敲除、分子生物学等技术，研究芸香科生物碱对肺上皮细胞EMT、COX2/PGE2以及可能下游信号通路分子的影响，进一步阐明其通过干预COX2/PGE2介导的EMT预防ARDS相关肺纤维化的机制。本研究为芸香科生物碱的抗纤维化提供新的理论依据，并为ARDS相关肺纤维化寻找可能的预防药物。

急性呼吸窘迫综合征(ARDS)是一种严重危及生命的临床危重疾病。ARDS相关的肺纤维化是ARDS常见的并发症。早期肺纤维化的发生表明多器官功能衰竭的发生率和死亡率较高。本研究建立了LPS诱导的小鼠ARDS相关肺纤维化模型。探讨芸香科生物碱（柑橘碱提取物，CAE）体内和体外抗肺纤维化作用及分子机制。通过HE染色、Masson染色、天狼星红染色和免疫组化等方法，证实CAE可改善肺损伤和肺纤维化。体外实验表明，CAE对E-钙粘蛋白（E-cadherin）和N-钙粘蛋白（N-cadherin）具有双重调控作用，这是上皮-间充质转化(EMT)的重要指标。进一步证明，CAE主要通过Wnt/β-catenin、Stat3/6和COX2/PGE2信号逆转TGF-β1诱导的EMT。进一步，采用RNA-seq技术探索CAE对基因表达的具体影响时，我们不仅发现TGF-β通路的转录水平变化、细胞粘附和分化的改变，也发现CAE也显著影响了金属离子、解剖发育和类固醇激素生物合成。这些结果提示了我们值得深入研究了解肺纤维化的发展过程和CAE的治疗靶点。因此，本研究为CAE抗纤维化提供了新的治疗作用，并为CAE治疗LPS诱导的肺纤维化提供了潜在的机制。