Neferine attenuates lipopolysaccharide-induced acute lung injury through AMP-activated protein kinase α1-dependent autophagy and reduced macrophage pyroptosis.

BACKGROUND

Excessive NOD-like receptor family pyrin domain-containing 3 (NLRP3) inflammasome activity is an important driver of inflammatory tissue damage in acute lung injury (ALI), yet effective targeted therapies remain lacking. Neferine, a natural bisbenzylisoquinoline alkaloid extracted primarily from the mature seed embryo of Nelumbo nucifera, has shown anti-inflammatory potential, but its role in inflammasome regulation during ALI is still not well understood.

PURPOSE: This study was designed to examine whether neferine could attenuate lipopolysaccharide (LPS)-induced ALI through modulation of NLRP3 inflammasome signaling and to explore the mechanism involved.

STUDY

DESIGN

We carried out a preclinical investigation using an LPS-triggered mouse model of ALI together with a macrophage pyroptosis model induced by LPS+nigericin (Nig). Both preventive administration and delayed post-challenge treatment with neferine were evaluated in vivo. To define pathway dependence, we combined pharmacologic AMP-activated protein kinase (AMPK) inhibition, Prkaa1 deficiency, and blockade of autophagic flux.

METHODS

Neferine was examined in LPS-treated mice and in macrophages exposed to LPS plus Nig. Lung pathology, inflammatory injury, and pyroptosis-related changes were assessed. Additional experiments focused on AMPK signaling, autophagic flux, and NLRP3 degradation.

RESULTS

Neferine attenuated inflammatory lung injury in mice, accompanied by reduced inflammatory cytokine production and decreased macrophage pyroptosis. Mechanistic analyses showed that neferine increased AMPK phosphorylation and downstream unc-51 like autophagy activating kinase 1 (ULK1) signaling, enhanced autophagic flux, and promoted K63-linked ubiquitination of NLRP3. These changes favored trafficking of NLRP3 into autophagic compartments and its subsequent lysosomal degradation, thereby limiting inflammasome activity. Pharmacological inhibition of AMPK signaling and genetic loss of Prkaa1 weakened the effects of neferine on ULK1-related autophagy and pyroptosis-associated readouts, supporting a functional contribution of AMPKα1 signaling to this process. Notably, neferine also remained effective when given after LPS exposure, reducing inflammatory injury and pyroptosis-associated readouts in the therapeutic setting.

CONCLUSION

These findings indicate that neferine alleviates inflammasome-related lung injury at least partly through AMPKα1-dependent autophagy, accompanied by increased ULK1 phosphorylation and subsequent autophagy-lysosomal clearance of NLRP3.