Caffeine suppresses GSDMD-associated pyroptotic responses in microglia with a PPARγ-related contribution and attenuates microglia-mediated OPC-like cell injury in vitro

To investigate whether caffeine mitigates microglia-mediated injury to OPC-like cells in vitro by suppressing GSDMD-associated pyroptotic responses and to assess the contribution of PPARγ-related regulation. Neonatal rat microglial RNA-seq (GSE126946) was analyzed by differential-expression and pathway-scoring approaches. Network pharmacology and molecular docking nominated PPARG as a candidate node. In HAPI microglia, siPPARγ knockdown was validated by RT-qPCR and Western blotting, and LPS/nigericin-induced pyroptotic responses were assessed after caffeine treatment. PPARγ, NLRP3, GSDMD-N, cleaved caspase-1, LDH, IL-1β, and conditioned-medium effects on CG4 cells were evaluated. PPARγ-status × caffeine interactions in the four model groups were tested by two-way repeated-measures ANOVA, with independent experiment as the matching factor. IH_LPS decreased the PPARγ score and increased the GSDMD-pyroptosis score. siPPARγ reduced PPARG mRNA and PPARγ protein to approximately 20% and 50% of siNC levels, respectively. Caffeine reduced NLRP3, GSDMD-N, cleaved caspase-1, LDH, and IL-1β in model microglia. Significant PPARγ-status × caffeine interactions were observed for NLRP3, GSDMD-N, cleaved caspase-1, IL-1β, and LDH. Sidak-adjusted comparisons showed significant caffeine-associated reductions in all five endpoints in the siNC background, whereas the corresponding simple effects did not reach statistical significance after PPARγ knockdown, consistent with modification of the caffeine response by PPARγ status. Caffeine-treated microglial conditioned medium improved CG4 viability and myelin-related expression, whereas these outcomes remained lower in conditioned-medium groups derived from PPARγ-knockdown microglia. Caffeine suppressed NLRP3/caspase-1/GSDMD-associated pyroptotic responses in microglia, while conditioned medium from caffeine-treated microglia was associated with improved OPC-like-cell outcomes in vitro. Significant PPARγ-status × caffeine interactions across molecular and functional pyroptosis-related endpoints support an important contribution of PPARγ-related regulation to the microglial response to caffeine. Together, these findings support a PPARγ-related contribution to the microglial response to caffeine, while the conditioned-medium findings link caffeine-associated changes in the microglial environment to improved downstream OPC-like-cell outcomes in vitro.