Nimbolide inhibits NLRP3 inflammasome activation via blocking NEK7-NLRP3 interaction and alleviates sepsis-induced lung injury.

BACKGROUND

Sepsis-associated acute lung injury (ALI) remains a critical clinical challenge with limited therapeutic options. The NLRP3 inflammasome drives pathological inflammation in ALI, yet clinical translation of existing inhibitors is hindered by toxicity. Natural products offer safer alternatives, but their mechanisms in targeting NLRP3 assembly are poorly defined.

METHODS

We screened a natural compound library for NLRP3 inhibitors using LPS/nigericin-stimulated murine peritoneal macrophages, measuring IL-1β release by ELISA. Mechanistic studies included immunoprecipitation (NEK7-NLRP3/ASC-NLRP3 interactions), ASC oligomerization/speck formation assays, DARTS, CETSA, and molecular docking. In vivo efficacy was evaluated in LPS-induced ALI and endotoxemia mouse models (histopathology, cytokine analysis, immunoblotting).

RESULTS

Nimbolide was identified as a potent NLRP3 inhibitor. It dose-dependently suppressed IL-1β secretion and caspase-1/GSDMD cleavage in murine/human (THP-1) macrophages, without affecting AIM2/NLRC4 inflammasomes or TNF-α production. Mechanistically, Nimbolide disrupted NEK7-NLRP3 binding and ASC oligomerization, thereby blocking NLRP3 inflammasome assembly. Further investigations revealed that Nimbolide enhanced the thermal stability of NLRP3 as demonstrated by the cell thermal shift assay (CETSA), conferred protease resistance as evidenced by the drug affinity responsive target stability (DARTS) assay, and exhibited high-affinity binding to NLRP3 with a binding energy of -7.62 kcal/mol through molecular docking studies. These results collectively suggest that Nimbolide can directly bind to NLRP3. In vivo, Nimbolide reduced pulmonary IL-1β levels, suppressed GSDMD cleavage, and attenuated lung injury pathology.

CONCLUSION

Nimbolide is a new natural inhibitor that selectively targets the NLRP3 interface and block NLRP3 inflammasome activation, offering a promising therapeutic strategy for NLRP3-driven inflammatory disorders like sepsis-associated ALI.