FOXO1胞核穿梭调控急性呼吸窘迫综合征肺泡ENaC的作用及机制

Pulmonary edema is one of the most important pathological features of acute respiratory distress syndrome (ARDS). The ability of alveolar fluid clearance (AFC) is critical to the prognosis of ARDS. Alveolar epithelial sodium channel (ENaC) is the rate-limiting step for AFC. To investigate the effective regulatory mechanisms of ENaC may be pivotal for the therapy of ARDS. In our previous research, we discovered that activation of PI3K-AKT/SGK1 signaling pathway within the lung could decrease the degradation of ENaC to leave more ENaC on the alveolar epithelial surfaces. However, except for the protein level, we also observed the increased transcriptional level of ENaC with obscure mechanism. As the downstream signal of PI3K-AKT, transcription factor FOXO1 plays important role in regulating the transcription of its target genes. Recently, the promoter of ENaC gene was found to contain the binding elements for FOXO1. Therefore, we speculate FOXO1 is probably the key factor involved in the regulation of ENaC mRNA by PI3K-AKT in alveolar epithelia. In this project, with gene transfection, RNA interference, signaling inhibitor, patch clamp and ARDS models, we intend to investigate the molecular mechanisms of PI3K-AKT-FOXO1 signaling pathway in the regulation of alveolar ENaC in the ARDS condition, which may help to find new potential therapeutic target for ARDS.

肺水肿是ARDS的重要病理特征之一，肺泡水肿液清除（AFC）能力直接关乎ARDS的预后。上皮钠通道（ENaC）是AFC的限速环节，探究有效的ENaC调控机制，可能成为治疗ARDS的关键。研究团队前期发现肺内激活PI3K-AKT/SGK1信号不仅能抑制ENaC降解从而增加其蛋白表达丰度,还可上调ENaC mRNA表达，但其转录调控机制尚不明晰。PI3K-AKT下游信号分子FOXO1在调控靶基因转录中发挥重要作用，而新近发现ENaC基因启动子包含FOXO1结合元件。据此我们推测FOXO1可能是PI3K介导肺泡ENaC转录上调的关键分子。本项目拟利用基因表达和干扰、信号节点抑制及膜片钳等方法，结合动物模型，研究FOXO1在PI3K-AKT的作用下对ARDS肺泡ENaC的转录调控机制，进一步完善ENaC的分子调控网络，为ARDS的治疗提供新的潜在靶点。

研究团队前期发现肺内激活PI3K-AKT/SGK1信号不仅能抑制ENaC降解从而增加其蛋白表达丰度,还可上调ENaC mRNA表达，但其在基因转录水平的调控机制尚不明晰。PI3K-AKT下游信号分子FOXO1在调控靶基因转录中发挥重要作用，而新近发现ENaC基因启动子包含FOXO1结合元件。据此我们推测FOXO1可能是PI3K介导肺泡ENaC转录上调的关键分子。本项目利用基因表达和干扰、信号节点抑制等方法，结合动物模型，研究FOXO1在PI3K-AK的作用下对ARDS肺泡ENaC的转录调控机制。研究发现，过表达持续活化的FOXO1（Thr24、 Ser256 和 Ser319位点突变）从转录水平显著降低ENaC的表达，而沉默FOXO1基因从转录水平显著增加ENaC的表达；胰岛素刺激可诱导FOXO1磷酸化并从转录水平显著增加ENaC的表达水平，而该效应可被PI3K/AKT抑制剂阻断。通过免疫荧光方法发现，基础状态下，FOXO1主要在肺泡上皮细胞核内表达，而胰岛素刺激后，FOXO1在胞浆内表达增加而胞核内表达明显减少，而预使用LY294002和沃曼青霉素后，该现象被明显抑制；过表达持续突变活化的FoxO1后，胰岛素诱导的FoxO1核-胞穿梭效应被显著抑制。通过动物实验发现，LPS诱导的急性肺损伤小鼠肺内FOXO1磷酸化水平和ENaC mRNA表达水平较对照组明显降低，胰岛素可促进FOXO1磷酸化及从转录水平增加ENaC表达，而PI3K/AKT抑制剂可阻断上述效应。上述研究结果提示，在肺泡上皮细胞中，FOXO1磷酸化与ENaC mRNA表达呈正相关；胰岛素可通过PI3K/AKT信号途径促进FOXO1磷酸化表达，从而从转录水平上调ENaC表达，对加速急性肺损伤时肺泡水肿液清除有一定的作用。