Bufei Yishen formula ameliorates chronic obstructive pulmonary disease by restoring alveolar macrophage efferocytosis...

Bufei Yishen formula (BYF) is a traditional Chinese medicine (TCM) prescription clinically used to improve pulmonary function and quality of life in patients with stable chronic obstructive pulmonary disease (COPD). However, its mechanism in regulating alveolar macrophage dysfunction remains unclear.

This study aimed to determine whether BYF ameliorates COPD by restoring alveolar macrophage efferocytosis and to elucidate the involvement of nuclear factor erythroid 2-related factor 2 (Nrf2) signaling.

Single-cell RNA sequencing (scRNA-seq) was used to characterize macrophage alterations in COPD. Cigarette smoke/Klebsiella pneumoniae-induced COPD animals and cigarette smoke extract (CSE) alveolar macrophages were employed to assess the effects of BYF on lung injury, inflammation, efferocytosis, oxidative stress, and Nrf2 signaling. Nrf2 involvement was verified with dimethyl fumarate and ML385, and Nrf2-binding constituents of BYF were identified using Affinity ultrafiltration-LC/MS (AU-LC/MS), molecular docking, and surface plasmon resonance (SPR).

scRNA-seq analysis revealed that impaired macrophage efferocytosis is closely associated with COPD progression, identifying defective apoptotic cell clearance as a key pathological process. Consistently, in COPD animal models, BYF markedly improved pulmonary function, attenuated emphysematous and inflammatory lung lesions, reduced pro-inflammatory cytokine production, and corrected the imbalance between matrix metalloproteinases and their tissue inhibitor. To further clarify the underlying mechanism, RNA sequencing (RNA-seq) profiling of alveolar macrophages showed that BYF-regulated genes were mainly enriched in macrophage efferocytosis and Nrf2-related pathways. Mechanistically, both in vivo and in vitro experiments demonstrated that BYF activated Nrf2 signaling, promoted downstream HO-1 expression, reduced oxidative stress, and consequently restored cigarette smoke-impaired alveolar macrophage efferocytosis. Importantly, pharmacological inhibition of Nrf2 largely abolished the protective effects of BYF, whereas Nrf2 activation reproduced similar benefits, confirming the essential role of Nrf2 signaling in BYF-mediated macrophage protection. Furthermore, integrated AU-LC/MS, molecular docking, and SPR analyses identified nine constituents in BYF that interact with Nrf2, among which ononin exhibited the highest affinity (KD = 20.4 μM). Finally, ononin was verified to promote Nrf2 nuclear translocation, activate Nrf2 downstream genes, and may contribute to BYF-mediated efferocytosis restoration.

BYF alleviates COPD-related lung injury, at least in part, by activating Nrf2 signaling and restoring alveolar macrophage efferocytosis. These findings provide mechanistic evidence supporting BYF as a potential therapeutic strategy for COPD.