Theaflavin alleviates thoracic aortic dissection by disrupting the interaction between microtubule affinity-regulating kinase 4 and NLRP3 to inhibit inflammasome assembly.

BACKGROUND

Thoracic aortic dissection (TAD) is a severe vascular disorder characterized by structural damage, where macrophage-driven inflammation and NLRP3 inflammasome activation play pivotal roles. While the broad anti-inflammatory properties of Theaflavin (TF) are known, its precise structural targets within the TAD cascade remain incompletely understood. We hypothesized that TF protects against TAD by competitively disrupting the MARK4-NLRP3 interaction, thereby suppressing macrophage pyroptosis and inflammasome assembly.

PURPOSE: To evaluate the therapeutic efficacy of TF in preventing the progression of TAD and to systematically elucidate its precise molecular and structural targets.

METHODS

A β-aminopropionitrile (BAPN)-induced murine TAD model was established to evaluate the efficacy of TF in mice. For Bone marrow-derived macrophages (BMDMs) and PMA-differentiated THP-1 macrophages, LPS and ATP stimulation was applied to activate the NLRP3 inflammasome. Transcriptomic sequencing, Western blot, immunofluorescence, co-immunoprecipitation (Co-IP), cellular thermal shift assay (CETSA), drug affinity responsive target stability (DARTS), and molecular docking were used to delineate TF's targets and mechanisms.

RESULTS

TF significantly reduced BAPN-induced mortality (from 40% to 15% at 20 mg/kg/d) and attenuated aortic dilation-decreasing the diameters of the ascending aorta, arch, and descending aorta by up to 17.0%, 29.2%, and 20.0%, respectively-by suppressing pro-inflammatory cytokines in mice. Mechanistically, TF directly bound to the NLRP3 NACHT domain (Gln359/Pro365 residues) to disrupt its interaction with MARK4, blocking NLRP3 trafficking to the microtubule organizing center (MTOC) and inflammasome assembly. TF also reduced GSDMD cleavage and pyroptosis, which was reversed by MARK4 overexpression.

CONCLUSION

TF attenuates TAD pathogenesis by structurally disrupting the spatial assembly of NLRP3, thereby inhibiting the MARK4/NLRP3/GSDMD pyroptosis axis in macrophages.