Abstract: Phosphodiesterase 4 is a key enzyme involved in the regulation of cell signal transduction, but its role in subarachnoid hemorrhage remains unclear. Neuronal pyroptosis has been reported to be involved in early brain injury after subarachnoid hemorrhage. This study aimed to investigate whether phosphodiesterase 4 contributes to early brain injury after subarachnoid hemorrhage by mediating neuronal pyroptosis and its related mechanisms. Endovascular perforation of male C57BL/6J mice was performed to model subarachnoid hemorrhage in vivo, and oxyhemoglobin was added to the culture medium of primary neurons to model subarachnoid hemorrhage in vitro. A phosphodiesterase 4-specific inhibitor, etazolate, was intraperitoneally injected 30 minutes after subarachnoid hemorrhage induction. Small interfering RNA (siRNA) was administered intracerebroventricularly 72 hours before subarachnoid hemorrhage to achieve genetic knockdown of phosphodiesterase 4. To investigate the mechanism, a nucleotide-binding oligomerization domain-like receptor pyrin domain containing 3 (NLRP3)-specific agonist, nigericin, was intracerebroventricularly injected 60 minutes before subarachnoid hemorrhage. Neuronal phosphodiesterase 4 expression increased after subarachnoid hemorrhage and reached the highest point at 24 hours. Etazolate treatment reduced neurological deficits and brain edema in mice, alleviated neuronal pyroptosis and inflammatory response, and improved neuronal injury. Treatment with phosphodiesterase 4 siRNA had the same neuroprotective effects as etazolate. Mechanistically, phosphodiesterase 4 triggered the nuclear factor kappa-B pathway, and simultaneously caused lysosomal and mitochondrial dysfunction after subarachnoid hemorrhage, which promoted NLRP3 inflammasome activation and induced neuronal pyroptosis. Blocking of phosphodiesterase 4 inhibited the nuclear factor kappa-B pathway, and improved lysosome and mitochondrial function. Activation of NLRP3 reversed the neuroprotective effects of etazolate without affecting phosphodiesterase 4 expression. Together, the results indicate that phosphodiesterase 4 regulates NLRP3-mediated neuronal pyroptosis in early brain injury after subarachnoid hemorrhage. Phosphodiesterase 4 may be a potential therapeutic molecular target for subarachnoid hemorrhage.

Key words: early brain injury, etazolate, lysosome function, mitochondrial function, neuron, nucleotide-binding oligomerization domain-like receptor pyrin domain containing 3 (NLRP3), nuclear factor kappa-B, phosphodiesterase 4, pyroptosis, subarachnoid hemorrhage