Betaine suppressed microglial pyroptosis in HMC3 cells. (A–D) HMC3 cells were treated with different concentrations Hcy, betaine or MCC950 and examined for cell viability. (E–F) Representative fluorescence images and intensity of ROS in HMC3 cells under different treatment groups, scale bar = 50 μm, 10× magnification. (G) Representative TEM images showing morphological changes of HMC3 cells under different treatments. (H) Release of LDH in HMC3 cells under different treatments. (I–J) Flow cytometry analysis of HMC3 cells pyroptosis in different treatment groups, stained by Annexin V-FITC and PI. (K–L) ELISA analysis of IL-18 and IL-1β levels in HMC3 cells. (M) The heat map showed the mRNA changes of NLRP3, ASC, pro-caspase-1, cleaved-caspase-1, GSDMD, GSDMD-N, IL-18 and IL-1β in HMC3 cells. (N–V) Western blot analysis of protein levels for NLRP3, ASC, pro-caspase-1, cleaved-caspase-1, GSDMD, GSDMD-N, IL-18 and IL-1β in HMC3 cells. Data were analyzed by one-way analysis of variance followed by multiple comparisons. Data are presented as mean ± SEM. *p < 0.05, **p < 0.01.