Novel NLRP3 inhibitors mitigate acute radiation-induced lung injury by suppressing pyroptosis in alveolar epithelial cells

Objective

Radiation-induced lung injury (RILI), a severe complication of thoracic tumor radiotherapy, is driven by NLRP3-mediated pyroptosis triggered by irradiation. This study aimed to design novel NLRP3 inhibitors targeting pyroptosis in alveolar epithelial cells to alleviate RILI and elucidate underlying mechanisms.

Methods

Quinoxalinone derivatives (QK-3A – QK-3E) targeting the NACHT domain of NLRP3 were synthesized. Their effects on the NLRP3/GSDMD pathway and cytokines (IL-1β, IL-18) were evaluated in irradiated A549 and MLE-12 cells using high-content Screening. A C57BL/6 mouse RILI model assessed compound efficacy via HE staining, Western blot, immunofluorescence. Safety was evaluated.

Results

Single-cell sequencing datasets analysis revealed a marked reduction in alveolar type 2 (AT2) cell proportion (from 12.41 % to 4.02 %) during early RILI, with pyroptosis-related genes showing heightened activity in AT2 cells. Molecular docking demonstrated QK-3E's strong binding to NLRP3 residues ARG351 and PHE575, inhibiting its activation. In vitro, QK-3D and QK-3E reduced radiation-induced cleaved-caspase-1/GSDMD-N expression(about 30 %–75 %), and IL-1β/IL-18 releases(about 30 %–50 %), suppressing AT2 pyroptosis. In vivo, QK-3D and QK-3E attenuated lung index elevation(about 25 %), mitigated pulmonary and systemic inflammation, and outperformed dexamethasone. They downregulated GSDMD-N(about 20 %-30) and cleaved-caspase-1(about 50 %–60 %) in SPC⁺ AT2 cells, blocking pyroptosis without cardiotoxicity, hepatotoxicity, or nephrotoxicity.

Conclusion

The NLRP3 inhibitors QK-3D and QK-3E effectively alleviate RILI by suppressing AT2 pyroptosis via NLRP3/GSDMD pathway inhibition, offering a potent and safe therapeutic candidate for early intervention. This study advances pyroptosis-targeted strategies for inflammatory diseases.