Notch1-TGFβ1通路经Snai1介导百草枯中毒肺间质纤维化中的上皮-间质转化

Paraquat is used for organic heterocyclic contact defoliants and herbicides in worldwide, which has strong toxicity to human and animals. But it has no effective antidotes,so the mortality is very high. Paraquat poisoning can cause irreversible late-onset pulmonary fibrosis, and the patient eventually died of respiratory failure. But the pathogenic mechanism is unclear. These years, epithelial-mesenchymal transition (EMT) become one of the potent factors. In this study, we will detect the changes of migration capacity and EMT hallmarkers of type II epithelial cells after Notch1 pathway induction, and the expression of TGF-β1 and Snai1. And further study of specific action sites for marker protein α-SMA promoter by Notch1, TGF-β1 and Snai1 is adopted to find the potential sites for EMT. On the basis, RNA interference technology is used to silent Notch1 pathway to demonstrate that Notch1-TGFβ1 signalling mediates pulmonary EMT caused by paraquat was throgh the induction of Snai1. This is the first investigation on Notch1-TGFβ1-Snai1 function on EMT in paraquat induced pulmonary EMT, which will provide basis on understanding mechanism of paraquat-induced pulmonary interstitial fibrosis, provide new thinking on paraquat poisoning and find new targets for the treatment of paraquat.

百草枯是目前广泛使用的除草剂，对人畜均有较强的毒性，无特效解毒剂，近年来死亡率居高不下。百草枯中毒后可引起不可逆的迟发型肺纤维化，患者最终死于呼吸衰竭，近年来肺上皮细胞向间质细胞转变成为研究的焦点之一。本研究中，我们将检测激活Notch1通路后引起肺上皮细胞迁移能力和EMT标记物的变化，以及在此过程中TGF-β1和Snai1的表达的变化。之后，我们将进一步分析Notch1、TGF-β1、Snai1对EMT标志蛋白α-SMA启动子的具体作用位点，寻找可能的作用元件。在此基础上，采用RNA干扰技术沉默大鼠体内Notch1通路，进一步证明在大鼠百草枯致肺间质纤维化过程中Notch1-TGFβ1对EMT的作用是通过Snai1介导的。这是首次在百草枯致肺间质纤维化模型中探讨Notch1-TGFβ1-Snai1对EMT的作用，为研究百草枯中毒提供了新的思路，为百草枯中毒的治疗寻找新的靶点。

百草枯是目前广泛使用的除草剂，对人畜均有较强的毒性，无特效解毒剂，近年来死亡率居高不下。百草枯中毒后可引起不可逆的迟发型肺纤维化，患者最终死于呼吸衰竭，近年来肺上皮细胞向间质细胞转变成为研究的焦点之一。本研究中，我们观察到小剂量长时间百草枯刺激致A549细胞EMT发生。我们采用Notch'1通路Jagged-1配体和Notch'1胞内活性结构域NICD激活Notch1 通路后，引起肺上皮细胞迁移能力和EMT 标记物的变化。在此过程中TGF-β 1/SMAD3和Snai1 的表达量增加。而Notch1通路抑制剂DAPT可以使EMT标记物表达量下降，细胞迁移能力下降。之后，我们进一步分析了Notch1、TGF-β 1、Snai1 对EMT 标志蛋白α-SMA 启动子的具体作用位点，上述因子都能使α-SMA启动子表达量增强。敲除CArG、TCE元件的α-SMA启动子荧光强度显著降低，证明CArG、TCE是影响该通路的主要元件。在此基础上，采用RNA 干扰技术沉默小鼠体内Notch1 通路，进一步证明在小鼠百草枯致肺间质纤维化过程中Notch1-TGF β 1 对EMT的作用。这是首次在百草枯致肺间质纤维化模型中探讨Notch1-TGFβ1-Snai1 对EMT 的作用，为研究百草枯中毒提供了新的思路，为百草枯中毒的治疗寻找新的靶点。