Remifentanil-induced inflammation in microglial cells: Activation of the PAK4-mediated NF-κB/NLRP3 pathway and onset of hyperalgesia

Background: The perioperative use of remifentanil is associated with postoperative hyperalgesia, which can impair recovery and extend hospitalization. Recent studies have revealed that microglia-mediated activation of the NLRP3 inflammasome plays a critical role in opioid-induced hyperalgesia, with NF-κB acting as a pivotal activation point for NLRP3. Despite these findings, the specific molecular mechanisms underlying remifentanil-induced postoperative hyperalgesia remain unclear. This study aims to develop a model of remifentanil-induced hyperalgesia and investigate the molecular mechanisms, focusing on the NF-κB/NLRP3 pathway, using both in vitro and in vivo approaches.

Method: We established a remifentanil-induced hyperalgesia model and performed proteomic analysis to identify differential protein expression in the spinal cord tissue of rats. NLRP3 or PAK4 antagonists were administered intrathecally in vivo, and mechanical pain thresholds in the hind paws were measured using Von Frey testing. In vitro, we applied NLRP3 or PAK4 inhibitors or used lentivirus Infection to silence PAK4, NF-κB, and NLRP3 genes. Protein expression was assessed through immunohistochemistry, immunofluorescence, and Western blotting. Additionally, ELISA was performed to measure IL-1β and IL-18 levels, and RT-qPCR was conducted to evaluate the transcription of target genes.

Results: Proteomic analysis revealed that remifentanil upregulates PAK4 protein in spinal cord tissue two hours after the surgery. In addition, remifentanil induces morphological changes in the spinal cord dorsal horn, characterized by increased expression of PAK4, p-p65, NLRP3 and Iba-1 proteins, which in turn leads to elevated IL-1β and IL-18 levels and an inflammatory response. Intrathecal injection of NLRP3 or PAK4 inhibitors mitigates remifentanil-induced hyperalgesia and associated changes. In vitro, downregulation of PAK4 inhibits the increase in PAK4, p-p65, NLRP3 and Caspase-1 induced by LPS. Conversely, the downregulation of NLRP3 does not impact the levels of PAK4 and p-p65 proteins, aligning with the in vivo results and suggesting that PAK4 acts as an upstream signaling molecule of NLRP3.

Conclusion: Remifentanil can increase PAK4 expression in spinal cord dorsal horn cells by activating the NF-κB/NLRP3 pathway and mediating microglial activation, thereby contributing to postoperative hyperalgesia.

Hyperalgesia; Inflammatory response; Microglial cells; NF-κB; NLRP3; PAK4; Remifentanil.