CK1α-PTEN信号轴在ARDS相关肺纤维化的关键作用研究

Mechanical ventilation is often necessary in the treatment of acute respiratory distress syndrome (ARDS) patients, but can induce or aggravate lung injury and promote lung fibrosis. Clinical evidence suggests that pulmonary fibrosis is associated with a poor outcome in patients with ARDS. Endothelial cells could transdifferentiate to mesenchymal cells under the pathological injury, a process termed endothelial mesenchymal transition (EndoMT) which involved in multiple organ fibrosis. Our previous study found that in vitro mechanical stretch induced EndoMT markers change in human pulmonary vascular endothelial cells and decreased expression of CK1α and PTEN; combined with our previous results that CK1α can directly bind ubiquitin proteasome degradation inhibition of PTEN and significantly prolonged the half life of PTEN. We hypothesize that: mechanical stretch induced the downregulation of CK1α and PTEN, thereby activating the AKT signaling pathway which mediate EndoMT and promote mechanical stress induced lung fibrosis. Thus, in this grant, To evaluate whether endothelial cells could represent an additional source for fibroblasts, mechanical ventilation induced lung fibrosis was established in Tg (TIE2-lacZ) transgenic mice transgenic mice, in which LacZ was stably expressed in endothelial cells. By over-expression of CK1α or PTEN, or inhibition of AKT activity in vitro and in vivo animal studies, we plan to study the EndoMT and ventilation-induced lung fibrosis was mediated through CK1α-PTEN signaling pathway.

机械通气是急性呼吸窘迫综合征（ARDS）患者救治的主要手段，但机械通气可以加重肺损伤和促进纤维化，ARDS患者出现肺纤维化提示预后不佳。内皮细胞在损伤后可发生间质细胞转化，是多种脏器纤维化的重要病理机制。我们预实验结果证实肺血管内皮细胞在体外机械牵张后发生间质转化并出现CK1α和PTEN表达下降。结合我们前期研究结果CK1α能够与PTEN发生直接结合抑制PTEN的泛素蛋白酶体降解，显著延长PTEN的半衰期，我们提出“机械牵张内皮细胞导致CK1α和PTEN表达下降，激活AKT信号通路介导内皮间质转化促进肺纤维化”的科学假说。采用前期建立的ARDS肺纤维化动物模型，使用内皮细胞特异表达LacZ的转基因小鼠证实内皮间质转化在机械通气肺纤维化中重要作用；体外细胞和体内动物实验中过表达CK1α/PTEN或抑制AKT活性，证实CK1α-PTEN信号轴调控内皮间质转化促进机械通气相关肺纤维化的作用。

急性呼吸窘迫综合征（ARDS）是重症监护室常见的呼吸危重症，机械通气是ARDS患者救治的重要手段，但机械通气本身可以加重肺损伤和促进肺纤维化的发生，前期研究证实，发生肺纤维化的ARDS患者预后不佳。本项目主要研究目标为阐述机械通气介导ARDS发生纤维化的作用和分子机制，为ARDS机械通气相关肺纤维化的药物治疗靶点提供科学依据。本项目（1）通过回顾性分析ARDS患者的临床资料，证实ARDS患者存在较高的肺纤维化发生率；（2）通过体外机械牵张肺血管内皮细胞实验，证实机械牵张可导致肺血管内皮通透性增高，内皮细胞屏障功能障碍和发生间质转分化；（3）通过构建盐酸气管灌注-机械通气双重打击的急性肺损伤和机械通气相关肺纤维化模型，证实内皮细胞间质转分化在ARDS机械通气相关肺纤维化中的作用；发现外源性过表达miR-126、CK1α或PTEN可减轻ARDS机械通气相关的内皮损伤、内皮屏障功能障碍和肺纤维化；（4）发现ARDS患者发生巨细胞活动性感染与肺纤维化的发生相切相关，并证实巨细胞活动性感染导致上皮细胞发生间质转分化促进ARDS患者肺纤维化的发生的作用和机制。本项目系统性通过ARDS患者肺纤维化发生率的临床研究、体外机械牵张内皮细胞模型和体内ARDS机械通气相关肺纤维化动物模型，阐明ARDS患者机械通气导致内皮细胞损伤、内皮屏障功能损伤和肺纤维化发生的关键作用和机制，为ARDS机械通气相关性肺纤维化的治疗提供了全新的靶点。通过项目的实施，共发表期刊论文13篇，其中SCI收录论文10篇，中文核心期刊3篇，完成了项目研究目标。