Targeting the smooth muscle cell Keap1-Nrf2-GSDMDpyroptosis axis by cryptotanshinone prevents abdominal aortic aneurysm formation

Rationale: Abdominal aortic aneurysm (AAA) is an inflammatory, fatal aortic disease that currently lacks any effective drugs. Cryptotanshinone (CTS) is a prominent and inexpensive bioactive substance derived from Salvia miltiorrhiza Bunge, a well-known medicinal herb for treating cardiovascular diseases through its potent anti-inflammatory properties. Nevertheless, the therapeutic effect of CTS on AAA formation remains unknown. Methods: To investigate the therapeutic effect of CTS in AAA, variety of experimental approaches were employed, majorly including AAA mouse model establishment, real-time polymerase chain reaction (PCR), RNA sequencing, western blot, co-immunoprecipitation, scanning/transmission electron microscopy (SEM/TEM), enzyme-linked immunosorbent assay (ELISA), seahorse analysis, immunohistochemistry, and confocal imaging. Results: In this study, we demonstrated that CTS suppressed the formation of AAA in apolipoprotein E knock-out (ApoE-/-) mice infused with Ang II. A combination of network pharmacology and whole transcriptome sequencing analysis indicated that activation of the Keap1-Nrf2 pathway and regulation of programmed cell death in vascular smooth muscle cells (VSMCs) are closely linked to the anti-AAA effect of CTS. Mechanistically, CTS promoted the transcription of Nrf2 target genes, particularly Hmox-1, which prevented the activation of NLRP3 and GSDMD-initiated pyroptosis in VSMCs, thereby mitigating VSMC inflammation and maintaining the VSMC contractile phenotype. Subsequently, by utilizing molecular docking, together with the cellular thermal shift assay (CETSA) and isothermal titration calorimetry (ITC), a particular binding site was established between CTS and Keap1 at Arg415. To confirm the binding site, site-directed mutagenesis was performed, which intriguingly showed that the Arg415 mutation eliminated the binding between CTS and the Keap1-Nrf2 protein and abrogated the antioxidant and anti-pyroptosis effects of CTS. Furthermore, VSMC-specific Nrf2 knockdown in mice dramatically reversed the protective action of CTS in AAA and the inhibitory effect of CTS on VSMC pyroptosis. Conclusion: Naturally derived CTS exhibits promising efficacy as a treatment drug for AAA through its targeting of the Keap1-Nrf2-GSDMD-pyroptosis axis in VSMCs.