Effusol ameliorates ischemic stroke by targeting NLRP3 protein to regulate NLRP3 inflammasome-mediated pyroptosis.

Background: The significance of pyroptosis as an inflammatory mode of death in ischemic stroke (IS) has attracted much attention in recent years. Effusol is a dihydrophenanthrene component extracted from Juncus effusus L.. Previous studies have found that Juncus effusus L. has a good inhibitory effect against microglia activation. However, it is not clear whether effusol inhibits microglia over-activation and attenuates its mediated microglia pyroptosis in the treatment of IS.

Purpose: The aim is to examine how effusol influences the initiation and activation stages of pyroptosis, as well as the NLRP3 inflammasome, resulting from microglial over-activation triggered post-IS.

Methods: This study investigated the impact of effusol on neurological severity and edema to assess its neuroprotective effects in IS. Mechanistically, immunofluorescence and western blotting were applied to explore the initiation and activation of the NLRP3 inflammasome. Finally, we employed the NLRP3 specific inhibitor, molecular docking, drug affinity responsive target stability (DARTS), and cellular thermal shift assay (CETSA) to further explore the underlying targets of effusol.

Results: Effusol mitigated IS-induced damage and downregulated the expression of inflammatory factors at the mRNA level, the protein levels of toll-like receptor 4 (TLR4), nuclear transcription factor NF-κB p65, and key components of the NLRP3 inflammasome. Effusol also mitigated mitochondrial damage by increasing ATP levels and decreasing mitochondrial membrane potential. Importantly, effusol targets NLRP3 protein to inhibit pyroptosis, thereby suppressing the hyperactivation of NLRP3 inflammasome.

Conclusions: Effusol may be protective against IS by targeting NLRP3 proteins to inhibit NLRP3 inflammasome activation-mediated pyroptosis. This finding provides a theoretical basis and a prospective drug candidate for the treatment of effusol in IS.