TLR4 aggravates microglial pyroptosis by promoting DDX3X‐mediated NLRP3 inflammasome activation via JAK2/STAT1 pathway after spinal cord injury

Abstract Background Toll‐like receptor 4 (TLR4) participates in the initiation of neuroinflammation in various neurological diseases, including central nervous system injuries. NLR family pyrin domain containing 3 (NLRP3) inflammasome‐mediated microglial pyroptosis is crucial for the inflammatory response during secondary spinal cord injury (SCI). However, the underlying mechanism by which TLR4 regulates NLRP3 inflammasome activation and microglial pyroptosis after SCI remains uncertain. Methods We established an in vivo mouse model of SCI using TLR4‐knockout (TLR4‐KO) and wild‐type (WT) mice. The levels of pyroptosis, tissue damage and neurological function recovery were evaluated in the three groups (Sham, SCI, SCI‐TLR4‐KO). To identify differentially expressed proteins, tandem mass tag (TMT)‐based proteomics was conducted using spinal cord tissue between TLR4‐KO and WT mice after SCI. For our in vitro model, mouse microglial BV2 cells were exposed to lipopolysaccharides (1 µg/ml, 8 h) and adenosine triphosphate (ATP) (5 mM, 2 h) to induce pyroptosis. A series of molecular biological experiments, including Western blot (WB), real‐time quantitative polymerase chain reaction (RT‐qPCR), enzyme‐linked immunosorbent assay (ELISA), immunofluorescence (IF), immunohistochemical (IHC), chromatin immunoprecipitation (ChIP), Dual‐Luciferase Reporter assay (DLA) and co‐immunoprecipitation (Co‐IP), were performed to explore the specific mechanism of microglial pyroptosis in vivo and in vitro. Results Our results indicated that TLR4 promoted the expression of dead‐box helicase 3 X‐linked (DDX3X), which mediated NLRP3 inflammasome activation and microglial pyroptosis after SCI. Further analysis revealed that TLR4 upregulated the DDX3X/NLRP3 axis by activating the JAK2/STAT1 signalling pathway, and importantly, STAT1 was identified as a transcription factor promoting DDX3X expression. In addition, we found that biglycan was increased after SCI and interacted with TLR4 to jointly regulate microglial pyroptosis through the JAK2/STAT1/DDX3X/NLRP3 axis after SCI. Conclusion Our study preliminarily identified a novel mechanism by which TLR4 regulates NLRP3 inflammasome‐mediated microglial pyroptosis in response to SCI—providing a novel and promising therapeutic target for SCI.