Wnt/β-catenin信号通路在百草枯中毒致肺纤维化中的作用研究

Paraquat (PQ) is a widely used herbicide in the world. Paraquat poisoning could lead to irreversible pulmonary fibrosis and lead to respiratory failure to cause death in patients finally. However, no specific antidote is used to treatment patients and the mortality is still high. Recent studies have found that paraquat poisoning could cause the excessive expression of cytokines, which finally lead to pulmonary fibrogenesis. Our previous study found that paraquat induced up-regulation of Wnt signaling expression in the lung tissue, and other studies have demonstrated that activated Wnt signaling pathway could promote the pulmonary fibrogenesis. This project based on our previous study to put forward to Wnt signaling pathway as the breakthrough point and carry out the following research. 1. Establish the pulmonary fibrosis model induced by intraperitoneal injection of paraquat in rats. Then verify that activated Wnt signaling is involved in the pulmonary fibrogenesis process, but blockade of Wnt signaling pathway would inhibit the pulmonary fibrosis. 2. Establish EMT (epithelial-mesenchymal transition) model induced by PQ in alveolar epithelial cells. Use antibody array to determine the key Wnt proteins involved in EMT process and design the specific siRNA of key Wnt proteins. 3. Use the specific siRNA of key Wnt proteins in treatment of EMT model and pulmonary fibrosis model of rats to clarify that specific inhibition of Wnt signaling pathway could inhibit pulmonary fibrogenesis and would be benefit to promote lung tissue repair. In the present study, we would screen the crucial Wnt proteins in the process of pulmonary fibrogenesis induced by paraquat. This study intends to provide experimental support and theoretical basis for searching the crucial molecular targets in treatment of paraquat-induced pulmonary fibrosis.

百草枯(PQ)是一种广泛使用的除草剂，PQ中毒患者致死的主要原因是不可逆的肺纤维化导致的呼吸衰竭。目前尚无特效解毒药，死亡率高。研究表明PQ中毒可引起肺组织内细胞因子过度表达，进而导致肺纤维化的发生发展。我们前期研究发现PQ导致肺组织Wnt信号通路高度激活，而多项研究证实激活的Wnt信号通路能够促进肺纤维化。本项目在前期工作基础上提出，以Wnt信号通路为切入点，开展以下研究：1.建立PQ中毒大鼠肺纤维化模型，验证Wnt信号通路的激活参与肺纤维化进程，阻断Wnt信号通路能够抑制肺纤维化；2.建立PQ诱导的EMT模型，采用蛋白芯片筛选参与EMT进程的关键Wnt蛋白，设计其特异性siRNA；3.在细胞模型和动物模型中验证特异性阻断Wnt信号通路能够抑制肺纤维化的发生发展，促进肺组织修复。本课题筛选参与PQ导致肺纤维化发生发展的关键Wnt蛋白，为寻找治疗PQ中毒肺纤维化的关键分子靶点提供理论依据。

百草枯（Paraquat, PQ）是一种目前仍广泛使用的有机杂环类除草剂，对人体毒性极大，其致死率高，临床治疗效果差，治愈率低。肺部是PQ主要的靶器官，多数患者死于不可逆的肺纤维化导致的呼吸衰竭。PQ致肺纤维化的发病机制目前尚不清楚，因此积极探究PQ致肺纤维化发生发展的分子机制，寻求抑制肺纤维化的有效途径是提高中毒患者存活率的关键。本研究从分子水平、细胞水平、动物水平揭示PQ致肺纤维化的具体分子机制，并寻找干预PQ致肺纤维化的关键分子靶点。通过腹腔注射PQ成功建立肺纤维化大鼠模型，发现PQ能够导致肺纤维化发生。同样地，PQ能够导致肺上皮细胞EMT发生。采用抗体芯片筛选发现Wnt信号通路中关键蛋白β-catenin和MMP-2在PQ中毒肺纤维化大鼠肺组织中高表达，并通过实验进行了验证，证明PQ能够导致Wnt信号通路高度激活，激活的Wnt信号通路可能参与了PQ导致的肺纤维化进程。为了验证Wnt信号通路在其中的作用，采用Wnt信号通路广谱抑制剂DKK1处理PQ诱导的肺上皮细胞EMT模型和肺纤维化动物模型，发现DKK1能够抑制PQ激活的Wnt信号通路，抑制PQ导致的EMT发生，抑制肺纤维化的发生发展。随后，采用基因芯片和Q-PCR检测PQ处理的肺上皮细胞胞EMT进程中表达显著变化的Wnt细胞因子，发现Wnt2，Wnt3a，Wnt5a，Wnt8a和Wnt10b基因表达水平显著升高，进一步验证发现Wnt3a和Wnt10b蛋白表达水平显著升高，在PQ中毒大鼠肺组织中Wnt3a和Wnt10b蛋白表达水平显著上调。采用Wnt3a和Wnt10b重组细胞因子处理肺上皮细胞，发现其能够促进肺上皮细胞EMT进程，证明Wnt3a和Wnt10b在PQ导致的EMT和肺纤维化进程中起着重要的调控作用。设计并构建Wnt10b的siRNA慢病毒，给予PQ诱导的肺上皮细胞EMT模型和肺纤维化大鼠模型，发现Wnt10b-siRNA能够抑制Wnt10b的表达，显著阻断PQ激活的Wnt信号通路，从而抑制PQ导致的肺上皮细胞EMT进程和肺纤维化的发生发展，促进肺组织修复，恢复肺功能。Wnt3a的siRNA慢病毒已构建，关于Wnt3a的siRNA在PQ致肺纤维化中的作用验证实验正在进行中。本研究证明Wnt3a和Wnt10b可以作为潜在的干预PQ致肺纤维化的关键分子靶点，为临床治疗PQ致肺纤维化提供了理论依据。