Abstract Background and objectives As a leading global health concern, chronic obstructive pulmonary disease (COPD) is predominantly driven by cigarette smoke (CS). The pathogenesis of COPD involves excessive apoptosis of bronchial epithelial cells triggered by CS, which promotes chronic inflammation and disease progression. The specific molecular mediators of this apoptotic response, however, remain incompletely characterized. Although Rab genes are crucial regulators of intracellular processes and their dysfunction has been associated with pulmonary diseases, their involvement in COPD is not well established. This study, therefore, focuses on defining the role and mechanism of Rab32 in CS-induced bronchial epithelial apoptosis and COPD development. Methods To identify COPD-associated genes, we analyzed mRNA microarray data from 30 COPD patients and 163 controls. Meanwhile, single-cell RNA sequencing (scRNA-seq) data from 31 controls and 24 COPD patients were analyzed to pinpoint key phenotypes and molecular regulators in COPD pathogenesis. Rab GTPase expression was subsequently validated in clinical lung tissues from COPD patients and healthy volunteers. Furthermore, we assessed Rab32 levels in smoker-derived cells, CS-exposed mice, and cigarette smoke extract (CSE)-treated BEAS-2B cells to investigate the impact of CS on Rab32. The functional role of Rab32 was explored using Rab32 knockout mice and Rab32-knockdown/overexpression cell lines. A combination of lung function tests, H&E staining, Western blotting, and flow cytometry was utilized to assess apoptosis and disease progression. Finally, the involvement of the reactive oxygen species (ROS)-Rab32 axis was investigated by treating BEAS-2B cells and mouse models with the antioxidant N-Acetyl-L-cysteine (NAC) in the context of CSE or H2O2 exposure. Results Rab32 emerged from scRNA-seq data as a crucial regulator of apoptosis in bronchial epithelial cells in COPD. Rab32 was significantly upregulated in the lungs of COPD patients, smoker-derived epithelial cells, and CS-exposed mice, as well as in CSE-treated BEAS-2B cells. Functional studies revealed that CSE promoted the Rab32-mediated mitochondrial translocation of BAX, leading to increased mitochondrial permeability, loss of mitochondrial membrane potential, and activation of the mitochondrial apoptosis pathway. Rab32 knockout suppressed bronchial epithelial apoptosis, reduced levels of the inflammatory cytokine IL-6 in bronchoalveolar lavage fluid, and alleviated lung function impairment and emphysema in CS-exposed mice. The antioxidant NAC effectively reversed Rab32 upregulation, apoptosis, and lung injury in CS-exposed models. Conclusions Rab32 upregulation upon CS exposure drives emphysema and inflammation in COPD, by mediating BAX-related apoptotic pathways and mitochondrial dysfunction in bronchial epithelial cells, positioning it as a compelling therapeutic target in COPD. This abstract is funded by: National Natural Science (82270039, 81970035)
Wu et al. (Fri,) studied this question.