Pyroptosis was also evaluated by measuring the levels of LDH release upon the different treatments. The results in Figure 7B illustrate that either ethanol or ATP treatments induces the release of cytoplasmic LDH in the cell medium. In all cases, pyroptosis was abrogated by Z-YVAD-FMK (a caspase-1 inhibitor) or Z-VAD-FMK or Mito-TEMPO treatments (Figures 7A,B). Notably, ATP, LPS or ethanol also induced a slight, yet significant increase in apoptosis (PI- cells and increased staining with Hoechst 33342 due to DNA condensation). Thus, the percentage of apoptosis was 3.2, 3.3, 2.9, and 3.3% upon treatment with ATP, LPS or ethanol (10 and 50 mM), respectively (Figure 7A). No significant or minimal changes in pyroptosis or apoptosis were observed in treated TLR4-KO astrocytes.
