TLR2 signalling triggers NLRP3 inflammasome-mediated microglial pyroptosis promotes neonatal hypoxic-ischemic brain...

Neonatal hypoxic-ischemic encephalopathy (HIE) causes severe neurological damage. The extensive cell death and subsequent inflammation observed in the brain following hypoxic-ischemic (HI) injury suggest that pyroptosis, an inflammation-associated form of programmed cell death, may play a significant role in HIE. This study used bioinformatics tools to identify key genes related to pyroptosis. Further experimental validation was conducted using a neonatal mouse HIE model and a classical microglial pyroptosis induction in vitro, followed by treatment with specific inhibitors or small interfering RNA. The results were validated using neurobehavioral testing, Western blot, qRT-PCR, immunofluorescence, and transmission electron microscopy. Tlr2 was identified as a target gene to be validated. In vivo validation experiments demonstrated that TLR2 mRNA and protein levels were significantly elevated following HI insult. Mice treated with the TLR2-specific inhibitor C29 exhibited improved motor and cognitive functions and reduced brain injury, tissue abnormalities, and neuronal apoptosis compared to the HI group. Further investigation revealed that TLR2 inhibition effectively suppressed NLRP3 inflammasome activation and GSDMD-associated pyroptosis-related proteins, reversing pyroptosis features. Additionally, TLR2 inhibition decreased Iba1 expression and patially reversed the morphological and pyroptosis protein levels changes of microglia in the cortex. In vitro validation experiments showed that TLR2 knockdown attenuated LPS-induced pyroptosis in the microglial cell line BV2, with changes in related proteins consistent with in vivo findings. Using bioinformatics methods, we identified TLR2 as a key pyroptosis-related gene in the context of HIE. Further experimental validation revealed that TLR2 participated in brain injury and neurological dysfunction by affecting NLRP3 inflammasome-mediated pyroptosis.