Glibenclamide Directly Prevents Neuroinflammation by Targeting SUR1-TRPM4-Mediated NLRP3 Inflammasome Activation In Microglia

Abstract

Glibenclamide (GLB) reduces brain edema and improves neurological outcome in animal experiments and preliminary clinical studies. Recent studies also suggested a strong anti-inflammatory effect of GLB, via inhibiting nucleotide-binding oligomerization domain-like receptor containing pyrin domain 3 (NLRP3) inflammasome activation. However, it remains unknown whether the anti-inflammatory effect of GLB is independent of its role in preventing brain edema, and how GLB inhibits the NLRP3 inflammasome is not fully understood. Sprague-Dawley male rats underwent 10-min asphyxial cardiac arrest and cardiopulmonary resuscitation or sham-operation. The Trpm4 siRNA and GLB were injected to block sulfonylurea receptor 1-transient receptor potential M4 (SUR1-TRPM4) channel in rats. Western blotting, quantitative real-time polymerase chain reaction, behavioral analysis, and histological examination were used to evaluate the role of GLB in preventing NLRP3-mediated neuroinflammation through inhibiting SUR1-TRPM4, and corresponding neuroprotective effect. To further explore the underlying mechanism, BV2 cells were subjected to lipopolysaccharides, or oxygen-glucose deprivation/reperfusion. Here, in rat model of cardiac arrest with brain edema combined with neuroinflammation, GLB significantly alleviated neurocognitive deficit and neuropathological damage, via the inhibition of microglial NLRP3 inflammasome activation by blocking SUR1-TRPM4. Of note, the above effects of GLB could be achieved by knockdown of Trpm4. In vitro under circumstance of eliminating distractions from brain edema, SUR1-TRPM4 and NLRP3 inflammasome were also activated in BV2 cells subjected to lipopolysaccharides, or oxygen-glucose deprivation/reperfusion, which could be blocked by GLB or 9-phenanthrol, a TRPM4 inhibitor. Importantly, activation of SUR1-TRPM4 in BV2 cells required the P2X7 receptor-mediated Ca²⁺ influx, which in turn magnified the K⁺ efflux via the Na⁺ influx-driven opening of K⁺ channels, leading to the NLRP3 inflammasome activation. These findings suggest that GLB has a direct anti-inflammatory neuroprotective effect independent of its role in preventing brain edema, through inhibition of SUR1-TRPM4 which amplifies K⁺ efflux and promotes NLRP3 inflammasome activation.

Keywords: Cardiac arrest/cardiopulmonary resuscitation; Glibenclamide; Microglia; NLRP3 inflammasome; SUR1-TRPM4.