GETomics based bulk-RNA sequencing revealed HIF-3a prevents COPD by inhibiting alveolar epithelial cell ferroptosis via the HIF-3a-GPx4 axis.

COPD patients are largely asymptomatic until later stages when prognosis is generally poor. Here, by shifting the focus forward to pre-COPD and smoking stages, we found the peak of reactive oxygen species (ROS) level and hypoxia character are enriched in pre-COPD samples, and hypoxia inducible factor (HIF)-3a is the key factor. Smoking induced regional tissue hypoxia and emphysema have been found in COPD patients. However, the mechanisms underlying hypoxia especially HIF-3a and COPD have not been investigated. In this study, by leveraging scRNA-seq and EpCAM co-localization analysis we identified HIF-3a is downregulated in alveolar epithelial cells in COPD. In vitro experiments using lentivirus transfection, bulk-RNA seq and RSL3 we found the activation of HIF-3a-GPx4 axis inhibits alveolar epithelial cell ferroptosis under the treatment of cigarettes smoking extracts (CSE). Further results from SftpcCreert2/+R26LSL-Hif3a knock-in mice demonstrated overexpression of HIF-3a inhibits alveolar epithelial cells ferroptosis and prevents the declining of lung function. Our data suggest the activation of HIF-3a-GPx4 axis prevents COPD by inhibiting alveolar epithelial cells ferroptosis. By leveraging this comprehensive analysis method, more promising treatment targets will be found and enhance our understanding to the pathogenesis. Overall design: To explore the decades long COPD pathogenesis process by including non-smokers, smokers, pre-COPD and COPD patients.Using transcriptomics technology to delineate the changing chracter.

慢性阻塞性肺疾病（Chronic Obstructive Pulmonary Disease, COPD）患者在疾病进展至晚期前大多无明显临床症状，此时预后通常较差。本研究将研究焦点前移至COPD前期与吸烟阶段，发现活性氧（reactive oxygen species, ROS）水平峰值与缺氧特征在COPD前期样本中显著富集，且缺氧诱导因子（hypoxia inducible factor, HIF）-3α为核心调控因子。

吸烟可诱导COPD患者出现局部组织缺氧及肺气肿，但缺氧相关分子机制尤其是HIF-3α与COPD的关联尚未得到系统研究。

本研究通过单细胞RNA测序（single-cell RNA sequencing, scRNA-seq）与上皮细胞黏附分子（Epithelial Cell Adhesion Molecule, EpCAM）共定位分析，证实COPD患者肺泡上皮细胞中HIF-3α表达水平下调。

本研究通过慢病毒转染、批量RNA测序及铁死亡诱导剂RSL3开展体外实验，结果显示在香烟烟雾提取物（cigarette smoking extracts, CSE）处理条件下，HIF-3α-谷胱甘肽过氧化物酶4（GPx4）轴的激活可有效抑制肺泡上皮细胞铁死亡。

进一步通过SftpcCreert2/+R26LSL-Hif3a敲入小鼠的体内实验证实，HIF-3α过表达可抑制肺泡上皮细胞铁死亡并延缓肺功能下降。

本研究数据表明，激活HIF-3α-GPx4轴可通过抑制肺泡上皮细胞铁死亡从而延缓COPD进展。通过本研究采用的综合分析策略，有望发现更多具有转化潜力的治疗靶点，加深我们对COPD发病机制的理解。

整体实验设计：通过纳入非吸烟者、吸烟者、COPD前期人群与COPD患者，探究COPD长达数十年的发病机制进程；并借助转录组学技术刻画疾病相关分子特征的动态变化。