INT-777 attenuates NLRP3-ASC inflammasome-mediated neuroinflammation via TGR5/cAMP/PKA signaling pathway after subarachnoid hemorrhage in rats

Background: Inflammasome-mediated neuroinflammation plays an important role in the pathogenesis of early brain injury (EBI) following subarachnoid hemorrhage (SAH). The activation of the TGR5 receptor has been shown to be neuroprotective in a variety of neurological diseases. This study aimed to investigate the effects of the specific synthetic TGR5 agonist, INT-777, in attenuating NLRP3-ASC inflammasome activation and reducing neuroinflammation after SAH. Methods: One hundred and eighty-four male Sprague Dawley rats were used. SAH was induced by the endovascular perforation. INT-777 was administered intranasally at 1 h after SAH induction. To elucidate the signaling pathway involved in the effect of INT-777 on inflammasome activation during EBI, TGR5 knockout CRISPR and PKA inhibitor H89 were administered intracerebroventricularly and intraperitoneally at 48 h and 1 h before SAH. The SAH grade, short- and long-term neurobehavioral assessments, brain water content, western blot, immunofluorescence staining, and Nissl staining were performed. Results: The expressions of endogenous TGR5, p-PKA, and NLRP3-ASC inflammasome were increased after SAH. INT-777 administration significantly decreased NLRP3-ASC inflammasome activation in microglia, reduced brain edema and neuroinflammation, leading to improved short-term neurobehavioral functions at 24 h after SAH. The administration of TGR5 CRISPR or PKA inhibitor (H89) abolished the anti-inflammation effects of INT-777, on NLRP3-ASC inflammasome, pro-inflammatory cytokines (IL-6, IL-1 beta, and TNF-alpha), and neutrophil infiltration at 24 h after SAH. Moreover, early administration of INT-777 attenuated neuronal degeneration in hippocampus on 28 d after SAH. Conclusions: INT-777 attenuated NLRP3-ASC inflammasome-dependent neuroinflammation in the EBI after SAH, partially via TGR5/cAMP/PKA signaling pathway. Early administration of INT-777 may serve as a potential therapeutic strategy for EBI management in the setting of SAH.