CTGF-Wnt信号途径调控肺成纤维细胞转分化参与百草枯致肺纤维化的分子机制研究

PQ poisoning could lead to irreversible pulmonary fibrosis and lead to respiratory failure, which is the main reason of mortality of paraquat (PQ) poisoning patients. The pathogenesis of PQ-induced pulmonary fibrosis remains unclear and lack of clinic treatment, which leads to high mortality. In our study, we found that PQ could induce the expressions of CTGF and some specific Wnt proteins up-regulated. Many studies have demonstrated that CTGF and Wnt proteins could activate Wnt signaling pathway via combining their receptors. Aberrant activated Wnt signaling pathway is involved in pulmonary fibrogenesis. Therefore, clarifying the role of CTGF-Wnt pathway in PQ-induced pulmonary fibrosis helps to search the effective intervention targets for pulmonary fibrosis induced by PQ. On the basis of preliminary work, the project is proposed to carry out the following research: 1. To ascertain the role of CTGF-Wnt pathways in PQ-induced pulmonary fibrogenesis. 2. To verify the key Wnt proteins participating in the pulmonary fibrogenesis induced by PQ. 3. In the fibroblasts trans-differentiation cell model and pulmonary fibrosis animal model, to verify that targeted inhibition CTGF-Wnt signaling pathway could inhibit lung fibroblasts transdifferentiation into myofibroblasts and inhibit PQ-induced pulmonary fibrosis, which would promote lung tissue repair. This project would clarify the role of CTGF-Wnt signaling pathway in PQ-induced pulmonary fibrosis, which would help us to better understand the molecular mechanisms involved in PQ-induced pulmonary fibrosis and will provide the theoretical significance and guiding value for clinical treatment.

百草枯(PQ)中毒患者主要死亡原因是PQ导致的肺纤维化所引起的呼吸衰竭，其发病机制尚不清楚，临床缺乏有效解毒药，死亡率高。我们发现，PQ能够导致CTGF、Wnt蛋白表达水平升高，多项研究证实CTGF、Wnt蛋白能够与相应受体结合激活Wnt信号通路，而激活Wnt信号通路促进肺纤维化发生发展。因此进一步阐明CTGF-Wnt信号途径在肺纤维化中的调控作用，有助于寻找PQ致肺纤维化的有效干预靶点。本课题拟在前期工作基础上，开展以下研究：1. 验证CTGF-Wnt信号途径在PQ致肺纤维化中的调控作用；2. 确定参与PQ致肺纤维化发生发展的关键Wnt蛋白；3.细胞模型和动物模型中验证靶向性抑制CTGF-Wnt信号途径能够抑制肺成纤维细胞转分化过程，抑制PQ导致的肺纤维化进程，促进肺组织修复。本课题阐明CTGF-Wnt信号途径在PQ致肺纤维化中的作用机制，对于临床治疗具有理论意义和指导价值。

百草枯（PQ）是全球农业生产中广泛使用的一种高效除草剂，对人体毒性极大，主要经呼吸道吸入、消化道和皮肤粘附吸收。由于百草枯中毒致死剂量极低（口服致死剂量40mg/kg），临床上缺乏有效解毒剂，中毒患者死亡率居高不下。PQ中毒的特征性病理改变是快速进展的弥漫性肺纤维化，最终导致呼吸衰竭是中毒患者死亡的主要原因。因此阐明PQ致肺纤维化的发生机制，探索控制PQ致肺纤维化发生发展的关键分子靶点、寻求有效的治疗手段，是急诊医学领域亟待解决的重要问题之一。本研究通过分子实验、细胞实验和动物实验揭示PQ致肺纤维化发生发展的分子机制，筛选寻找抑制肺纤维化发生发展、促进肺组织修复的关键分子靶点，为临床治疗提供实验基础。.本研究建立PQ诱导的肺成纤维细胞转分化模型和PQ致肺纤维化大鼠模型，通过体内和体外实验发现，PQ致肺纤维化进程中，CTGF表达水平显著升高，Wnt/β-catenin信号通路高度激活。DKK1能够通过阻断Wnt/β-catenin信号通路，抑制PQ诱导的肺成纤维细胞向肌成纤维细胞转分化，抑制PQ导致的肺纤维化的发生发展，促进肺组织修复。体外实验证实CTGF能够促进肺成纤维细胞增殖，迁移，并转分化为肌成纤维细胞，从而参与PQ诱导的肺纤维化的发生发展。同时发现，CTGF能够促进肺成纤维细胞内Wnt/β-catenin信号通路的激活。构建CTGF的siRNA慢病毒载体，体内和体外实验证实抑制CTGF的表达水平能够阻断Wnt/β-catenin信号通路，抑制肺成纤维细胞向肌成纤维细胞转分化，抑制PQ导致的肺纤维化的发生发展。通过mRNA芯片检测结合Q-PCR和Western Blot实验验证发现Wnt1，Wnt3a，Wnt8b，Wnt10b可能是参与PQ致肺纤维化发生发展的关键Wnt蛋白，并通过细胞实验验证了Wnt3a能够激活Wnt/β-catenin信号通路促进肺成纤维细胞转分化。通过体内和体外实验证实Wnt3a的siRNA慢病毒能够显著抑制PQ导致的Wnt/β-catenin信号通路的激活，抑制肺成纤维细胞转分化，抑制PQ导致的肺纤维化的发生发展，促进肺组织修复。研究证实CTGF-Wnt信号途径是参与PQ致肺纤维化的发生发展的重要信号通路，CTGF和Wnt3a可作为干预PQ致肺纤维化的关键分子靶点。