Modulating inflammasome (NLRP3) activation and stress granule (SG) formation: Insight of neuroprotection by...

NBO therapy has demonstrated a neuroprotective effect on ischemic stroke. This study investigated the role of HIF-1α in regulating SG formation and NLRP3 inflammasome activation following I/R injury in NBO-induced neuroprotection.

A total of 137 adult male SD rats underwent 2 h of MCAO, followed by 2, 6, 24 or 48 h of reperfusion. NBO (95% O₂ at 2 l/min) was administered for 2 h at the onset of reperfusion. HIF-1α inhibitor (YC-1) was administered 2 h before MCAO. Brain damage was assessed by infarct volumes (TTC staining), LDH and ROS levels (ELISA), and apoptotic and pyroptosis cell death (flow cytometry and TUNEL assay). Gene and protein levels of HIF-1α and inflammasome related factors (IL-18, IL-1β, NLRP3, cleaved-Caspase-1, GSDMD-N, ASC, TXNIP) were analyzed. SG proteins levels (G3BP1, TIA-1) and DDX3X were detected by Western blot. Co-IP detected the interaction between DDX3X and G3BP1 or NLRP3.

Infarct volume, LDH expression, ROS levels, and cell death (apoptosis and pyroptosis) were significantly increased after I/R injury. NBO and YC-1 treatments significantly reduced infarct volume, LDH and ROS levels, and cell death at 24 and 48 h of reperfusion. NBO suppressed the expression of inflammasome-related markers (IL-1β, IL-18, NLRP3, TXNIP, ASC, cleaved-Caspase-1, GSDMD-N) at both mRNA and protein levels. Co-IP analysis showed that I/R enhanced the interaction between DDX3X and NLRP3, which was suppressed by NBO and YC-1. NBO increased SG formation by regulating G3BP1 and TIA-1 expression and strengthened the interaction between DDX3X and G3BP1. NBO + YC-1 did not show additive effects, indicating that the two treatments act through the same HIF-1α-dependent pathway.

NBO exerts strong neuroprotection against ischemic stroke by inhibiting HIF-1α-mediated NLRP3 inflammasome activation and enhancing SG formation via DDX3X-G3BP1 interaction. This study identifies HIF-1α as a key mediator of post-ischemic inflammation and stress response, highlighting NBO as a potent, mechanism-based therapeutic in ischemic stroke.