Homocysteine-Mediated Neuronal Pyroptosis Contributes to Brain Injury in Heatstroke Rats by Activating the m6A-YTHDF2-NLRP3 Pathway

Heat stroke (HS) is a life-threatening condition that leads to neuronal injury, particularly in the prefrontal cortex, though its mechanisms remain unclear. In this study, we established a rat HS model and observed significant inflammatory responses and neuronal pyroptosis in the prefrontal cortex 6 h post-heat exposure, with the injury severity increasing over time. Mechanistically, HS activated the caspase-1/GSDMD-dependent pyroptosis pathway through NLRP3 inflammasome activation, resulting in IL-1β and IL-18 release. Additionally, HS caused a marked increase in homocysteine (Hcy) levels in both the serum and the prefrontal cortex, accompanied by reduced expression of the Hcy metabolic enzymes MTHFR and CSE, suggesting Hcy metabolism disruption. In vitro, Hcy induced pyroptosis in PC12 cells, elevating IL-1β, IL-18, and LDH levels. Notably, the NLRP3 inhibitor MCC950 mitigated this effect by reducing IL-18 and LDH release. Reducing Hcy in vivo alleviated neuronal pyroptosis and counteracted the YTHDF2-mediated decrease in NLRP3 mRNA m⁶A modification. Hcy reduced global m⁶A modification, YTHDF2 expression, and NLRP3 m⁶A modification in PC12 cells. This study reveals that the activation of a novel m⁶A-YTHDF2-NLRP3 pathway by Hcy underlies HS-induced neuronal injury, suggesting potential therapeutic targets for HS intervention.