Chronic obstructive pulmonary disease (COPD) is an important public health challenge worldwide, and is usually caused by significant exposure to noxious agents, particularly cigarette smoke. Recent studies have revealed that excessive production of neutrophil extracellular traps (NETs) in the airways is associated with disease severity in COPD patients. NETs are extracellular neutrophil-derived structures composed of chromatin fibers decorated with histones and granule proteases including neutrophil elastase (NE). However, the effective prevention of NET formation in COPD remains elusive. Here, we demonstrated that treatment with GW311616A, a potent and selective inhibitor of NE, prevented cigarette smoke extract (CSE)-induced NET formation in human neutrophils by blocking NE nuclear translocation and subsequent chromatin decondensation. Inhibition of NE also abrogated CSE-induced ROS production and migration impairment of neutrophils. Administration of GW311616A in vivo substantially reduced pulmonary generation of NETs while attenuating the key pathological changes in COPD, including airway leukocyte infiltration, mucus-secreting goblet cell hyperplasia, and emphysema-like alveolar destruction in a mouse model of COPD induced by chronic cigarette smoke exposure. Mice treated with GW311616A also showed significant attenuation of neutrophil numbers and percentages and the levels of neutrophil chemotactic factors (LTB4, KC, and CXCL5) and proinflammatory cytokines (IL-1β, and TNF-α) in bronchoalveolar lavage fluid compared to mice treated with cigarette smoke exposure only. Furthermore, GW311616A treatment considerably improved lung function in the COPD mouse model, including preventing the decline of FEV₁₀₀/FVC and delta PEF as well as inhibiting the increase in FRC, TLC, and FRC/TLC. Overall, our study suggests that NE plays a critical role in cigarette smoke-induced NET formation by neutrophils and that inhibition of NE is a promising strategy to suppress NET-mediated pathophysiological changes in COPD.

Chronic obstructive pulmonary disease (COPD) imposes a significant global health and socioeconomic burden. Exacerbations of COPD (ECOPD), characterized by heightened airway inflammation and mucus hypersecretion, adversely affect patient health and accelerate disease progression. Qingke Pingchuan (QKPC) granules, a formulation from Traditional Chinese Medicine initially prescribed for acute bronchitis, have shown unexplored potential in ECOPD management, with mechanisms of action yet to be clarified. This study investigates the therapeutic effects of QKPC in a mouse model of ECOPD, focusing on underlying molecular mechanisms. COPD was induced in mice through chronic cigarette smoke (CS) exposure, followed by intratracheal administration of Pseudomonas aeruginosa lipopolysaccharide (LPS) to trigger exacerbation, after which mice were treated with QKPC granules. Major compounds in QKPC were identified via UHPLC-QE-MS, and high-throughput RNA sequencing of lung tissue samples identified differentially expressed genes. Transcriptomic data were integrated with network pharmacology analysis to pinpoint potential pathways, bioactive compounds, and target genes through which QKPC might attenuate ECOPD. Molecular docking, protein-small molecule binding assays, and in vitro analyses further validated interactions between key compounds and target genes, shedding light on plausible signaling pathways. QKPC treatment led to significant reductions in airway leukocyte infiltration and goblet cell metaplasia in CS- and LPS-exposed mice, accompanied by decreased levels of inflammatory cytokines (IL-6, IL-1β, CXCL1, and TNF-α) and mucin MUC5AC in bronchoalveolar lavage fluid. The integrative transcriptomic and network pharmacology analysis identified the neutrophil extracellular trap (NET) formation pathway as a key mechanism underlying QKPC's protective effect against ECOPD. In vitro assays demonstrated that epigallocatechin-3-gallate (EGCG) and quercetin, two important bioactive compounds in QKPC, significantly inhibited NETosis induced by cigarette smoke extract (CSE) plus LPS in human neutrophils. The two compounds were found to interact directly with the reactive oxidative species (ROS)-generating enzyme NOX2 and its regulatory subunit p47phox. Subsequent in vitro studies further confirmed EGCG and quercetin's capacity to reduce ROS production and downregulate NOX2 and p47phox protein levels in neutrophils stimulated with CSE and LPS. Additionally, in vivo studies confirmed QKPC's efficacy in reducing NET formation, oxidative stress, and NOX2/p47phox protein expression in the lung tissue of ECOPD mice. These findings suggest that QKPC granules alleviate airway inflammation in ECOPD, potentially through inhibition of pulmonary NET formation via the NOX2/p47phox-ROS pathway, underscoring their potential therapeutic application for ECOPD management in clinical settings.